Estrenes for inducing hypothalamic effects

ABSTRACT

The invention relates to estrene steroids, which bind to neuroepithelial receptors. The steroid is preferably administered in the form of a pharmaceutical composition containing one or more pharmaceutically acceptable carriers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 08/316,050, filed Sep. 29,1994, abandoned which is a continuation-in-part of application Ser. No.08/127,980 filed Sep. 28, 1993 now U.S. Pat. No. 5,783,571, which is acontinuation-in-part of U.S. patent application Ser. No. 07/903,525,filed Jun. 24, 1992 abandoned, which in turn is a continuation-in-partof U.S. patent application Ser. No. 07/707,862, filed May, 31, 1991abandoned, which in turn is a continuation-in-part of U.S. patentapplication Ser. No. 07/638,743, filed Jan. 7, 1991 and now abandoned.

The application also relates to another continuation-in-part of U.S.patent application Ser. No. 07/903,525, U.S. patent application Ser. No.08/077,140, filed Jun. 15, 1993, and to commonly assigned, co-pendingU.S. patent application Ser. No. 07/903,604, filed Jun., 24, 1991, (acontinuation-in-part of U.S. patent application Ser. No. 07/708,936,filed May 31, 1991, which in turn is a continuation-in-part of07/638,185, filed Jan. 7, 1991 and now abandoned) entitled "AndrosteneSteroids as Neurochemical Initiators of Change in Human HypothalamicFunction and Related Pharmaceutical compositions and Methods"; and tothe commonly assigned, co-pending continuation-in-part of 07/903,604,filed Jun. 24, 1992 U.S. Pat. application Ser. No. 08/077,359. Theaforementioned U.S. patent applications are each incorporated herein byreference.

Finally, this application may relate to a co-pending U.S. PatentApplication entitled "Frairance Compositions Containing HumanPheromones", filed Mar. 24, 1992, U.S. application Ser. No. 07/856,435.

TECHNICAL FIELD

This invention relates generally to novel compounds and methods foreffectuating change in human hypothalamic function, thereby alteringcertain behavior and physiology mediated by the hypothalamus ofindividuals. More particularly, the invention relates to the use ofcertain Estrene steroids as neurochemical effectuators of physiology andbehavior.

DESCRIPTION OF THE RELATED ART

The present invention relates to certain compounds, namely Estrenesteroids and related compounds as will be described herein, and methodsof using these compounds as human semiochemicals in order to alterhypothalamic function, thereby affecting certain consequent behavior andphysiology, e.g. the reduction of anxiety. Estrene steroids are typifiedby 17β-Estradiol (1,3,5(10)-Estratriene-3,17β-diol), and arecharacterized by a phenolic 1,3,5(10) A-ring and a hydroxy or hydroxyderivative, such as an ether or ester, at the 3-position. The pheromoneproperties of some Estrene steroids for some mammalian species has beendescribed. Michael, R. P. et al., Nature (1968) 218:746 refers toEstrogens (particularly Estradiol) as a pheromonal attractant of malerhesus monkeys. Parrot, R. F., Hormones and Behavior (1976) 7:207-215,reports Estradiol benzoate injection induces mating behavior inovariectomized rats; and the role of the blood level of Estradiol inmake sexual response (Phoenix, C. H., Physiol. and Behavior (1976)16:305-310) and female sexual response (Phoenix, C. H., Hormones andBehavior (1977) 8:356-362) in Rhesus monkeys has been described. On theother hand, there is little agreement in the literature as to whether ornot pheromones as such play any role in the reproductive behavior andinterpersonal communication of mammals (Beauchamp, G. K., et al., "ThePheromone Concept in Mammalian Chemical Communication: A Critique", In:Mammalian Olfaction, Reproductive Processes, and Behavior, Doty, R. L.,Ed., Academic Press, 1976).

An embodiment of the subject invention concerns the non-systemic, nasaladministration of certain Estrene steroids to affect a specificbehavioral or physiological response in human subjects, e.g. a reductionof negative affect, mood, and character traits. In particular, nasaladministration provides for contacting neuroreceptors of a heretoforepoorly understood neuroendocrine structure, commonly known as thevomeronasal organ ("VNO"; also known as "Jacobson's organ"), with one ormore steroid(s) or with compositions containing the steroid(s). Thisorgan is accessed through the nostrils of most higher animals--fromsnakes to humans, and has been associated, inter alia, with pheromonereception in certain species (see generally Muller-Schwarze &Silverstein, Chemical Signals, Plenum Press, N.Y. (1980)). The axons ofthe neuroepithelia of the vomeronasal organ, located supra palatial,form the vomeronasal nerve and have direct synaptic connection to theaccessory olfactory bulb and indirect input from there to thecorticomedial amygdaloid forebrain and hypothalamic nuclei of the brain.The distal axons of the terminal is nerve neurons may also serve asneurochemical receptors in the VNO. Stensaas, L. J., et al., J. SteroidBiochem. and Molec. Biol. (1991) 39:553. This nerve has direct synapticconnection with the hypothalamus.

Johnson, A. et al. (J. Otolaryngology (1985) 14:71-79) report evidencefor the presence of the vomeronasal organ in most adult humans, butconclude that the organ is probably non-functional. Contravening resultswhich suggest that the VNO is a functional chemosensory receptor arereported by Stensaas, L., et al., supra; and by Moran, D. T., et al.,Garcia-Velasco, J. and M. Mondragon; Monti-Bloch, L. and B. Grosser--allin J. Steroid Biochem. and Molec. Biol. (1991) 39.

It is apparent that it would be desirable to identify and synthesizehuman semiochemicals and pheromones and to develop pharmaceuticalcompositions and methods of use to influence hypothalamic function. Thisinvention relates to the unexpected discovery that, when nasallyadministered to human subjects, certain neurochemical ligands,particularly certain Estrene steroids and related compounds, orpharmaceutical compositions containing certain Estrenes or relatedcompounds, specifically bind to chemoreceptors of nasal neuroepithelialcells and this binding generates a series of neurolophysiologicalresponses resulting in an alteration of hypothalamic function of anindividual. When properly administered, the effect of certain of thesecompounds on the hypothalamus affects the function of the autonomicnervous system and a variety of behavioral or physiological phenomenawhich include, but are not limited to the following: anxiety,premenstrual stress, fear, aggression, hunger, blood pressure, and otherbehavioral and physiological functions normally regulated by thehypothalamus. Otto Appenzeller. The Autonomic Nervous System. Anintroduction of basic and clinical concepts (1990); Korner, P. I.Central nervous control of autonomic cardiovascular function, and Levy,N. M. and Martin, P. J. Neural control of the heart, both in Handbook ofPhysiology; Section 2: Cardiovascular System--the heart, Vol I,Washington D.C., 1979, American Physiological Society; Fishman, A. P.,et al. editors, Handbook of Physiology. Section 3: Respiratory System.Vol. II. Control of breathing. Bethesda Md. 1986. American PhysiologicalSociety.

In some instances a single Estrene steroid, or related compound, isadministered, in some instances combinations of Estrene steroids and/orrelated compounds are administered.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide novel steroidswhich are human semiochemicals or pheromones and are suitable for nasaladministration in an individual.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

Objects of this invention are achieved by steroids with the formula:##STR1## wherein R₁ is selected from the group consisting essentially ofone or two hydrogen atoms, methyl, methylene, and one or two halo atoms;R₂ is absent or is selected from the group consisting essentially ofhydrogen and methyl; R₃ is selected from the group consistingessentially of oxo, hydroxy, lower alkoxy, lower acyloxy, benzoyl,cypionyl, glucuronide and sulfonyl; R₄ is selected from the groupconsisting essentially of hydrogen, hydroxy, lower alkoxy, loweracyloxy, oxo and halo; R₅ is absent or is selected from the groupconsisting essentially of hydrogen, hydroxy, lower alkoxy and loweracyloxy; R₆ is a hydrogen or a halo; and "a" represents optionalaromatic unsaturation of ring A of said steroid, or "b", "c", and "d"are each optional double bonds; and "e", "f", "g", "h", "i" and "j" areeach optional double bonds. In this embodiment, the steroid ispreferrably administered in the form of a pharmaceutical compositioncontaining one or more pharmaceutically acceptable carriers; with theprovisos that:

(I) when "a" is present, R₃ is hydroxy, and "j", "i", "g" and "h" areall absent, then (a) R₄ cannot be hydrogen; or (b) R₄ cannot be oxo if"e" and "f" are absent;

(II) when "a" is present, R₃ is hydroxy; and "j", "i" and "g" are allabsent, "h" is present, then R₁ cannot be methylene;

(III) when "a", "h" and "i" are present, (a) then at least one of "e" or"f" is present, (b) R₁ is methylene, or (c) R₁ is not hydrogen.

(IV) when "b" is present, R₃ is oxo, "g", "h", "i", "j" are all absent,R₅ is hydrogen, (a) then R₁ cannot be one or two hydrogens if "f" isabsent, or (b) if "f" is present R₁ cannot be methyl;

(V) when "b" and "j" are present and R₃ is oxo, hen at least one of "e"or "f" must be present or R₁ must be methylene;

(VI) when "c" is present, "d" is absent and R₃ is hydroxy, then (a) atleast "e" or "f" must be present, or (b) R₁ must be methylene;

(VII) when "c" and "d" are present and R₃ is methoxy, then (a) at least"e" or "f" must be present or (b) R₁ must be methylene;

(VIII) when "b" is present, R₃ is hydroxy and R₅ is hydrogen, then (a)at least "e" or "i" must be present or (b) R₁ must be methylene.

A preferred class of compounds are those in which "a" is present and"g", "h" or "i" are optional double bonds. Another preferred classcontains "b", "c" or "j" as a double bond. Yet another class contains"c" and "d" as double bonds. Still another class contains R₂ as methyland "e" as a double bond.

The term lower alkyl, lower alkoxy, etc., encompasses carbon chains of 1to 6 carbon atoms, preferrably 1 to 4 carbon atoms. Halo includes I, Br,F and Cl.

The steroids of the invention alter hypothalamic function and autonomicfunction in an individual. A ligand for a chemoreceptor displayed on thesurface of a nasal neuroepithelial cell is provided wherein the cell isa part of tissue other than olfactory epithelia; and, the ligand isadministered within a nasal passage of the individual such that theligand binds specifically to the chemoreceptor, resulting in analteration of hypothalamic function of the individual.

All embodiments of this application relate to and include the functionalequivalents of the steroid structures disclosed in these embodiments andto those modified steroids which demonstrate said functionalequivalence, whether or not the modified steroids are explicitlydisclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the synthesis of 1, 3 ,5 (10), 16-Estratetraen-3-ol.

FIGS. 2A, 2B, and 2C are graphic representations of theelectrophysiological effect on receptor potential of the localizedadministration of particular steroids to the vomeronasal organ of malesubjects (FIG. 2A) and to the olfactory epithelium (FIG. 2C). FIG. 2B isa graphic comparison of the effect of an Estrene on the VNO receptorpotential of male and female subjects.

FIG. 3 is a graphic representation of the electrophysiological effect ofthe localized administration of particular steroids to the vomeronasalorgan of male (3A) and female (3B) subjects.

FIG. 4 depicts various autonomic responses of male subjects to1,3,5(10), 16-Estratetraen-3-yl acetate. A=receptor potential of thevomeronasal neuroepithelium; B=change in galvanic skin response(K-ohms); C=change in skin temperature (degrees C.).

FIG. 5 depicts comparative changes in potential of the VNO afterexposure to the methyl ether and the acetate of1,3,5(10),16-Estratetraen-3-ol.

FIG. 6 depicts sexual dimorphism in local and autonomic responses to thestimulation of the VNO with vomeropherins. Various vomeropherins (200fmoles) and the diluent control were administered to 30 male and 30female subjects (ages 20 to 45 ) as described. Bars indicate the meanresponse of the population.

FIGS. 6A & B: EVG responses were measured as described in male (A) andfemale (B) subjects.

FIGS. 6C & D: Electrodermal activity was measured as described. Changes(measured in xΩ) in response due to delivery of vomeropherins to the VNOof each subject are shown in male (C) and female (D) subjects.

FIGS. 6E & F: Alpha-cortical activity was measured as described. Changesin response due to delivery of vomeropherins to the VNO of male (E) andfemale (F) subjects.

FIGS. 6G & H: Skin temperature (ST) was measured as described. Changesin response due to delivery of vomeropherins to the VNO of each subjectare shown in male (G) and female (H) subjects.

A=1, 3, 5(10),16-Estratetraen-3-yl acetate

B=Androsta-4,16-dien-3-one

C=1,3,5(10),16-Estratetraen-3-ol

D=3-Methoxy-Estra-1,3,5(10),16-tetraene

E=Androsta-4,16-dien-3α-ol

F=Androsta- 4,16-dien-3β-ol

FIG. 7 depicts electro-olfactgrams of male and female subjects inducedby stimulation of the OE with olfactants and vomeropherins A: 400 fmolesof the olfactants 1-carvone and cineole as well as 200 fmoles of thevomeropherins A, B, C, D and F; and the stereoisomer E were appliedseparately as one second pulses to the OE of 20 subjects (both male andfemale) and each EOG response was recorded as described. The olfactantsas well as E and B produced significant (p<0.01) local response. B: 400fmoles of the olfactants 1-carvone and cineole do not induce asignificant EVG response when delivered to the VNO of male and femalesubjects.

FIG. 8 depicts the electrophysiological effect of the followingvomeropherins on the vomeronasal organ of 20 female subjects:

    ______________________________________                                        G = Androst-4-en-3-one                                                          H = Androsta-4,16-diene-3,6-dione                                             J = 10,17-Dimethylgona-4,13(17)-dien-3-one                                    K = 1,3,5(10),16-Estratetraen-3-ol-methyl ether                               L = 1,3,5(10),16-Estratetraen-3-yl-propionate                                 EVG = Electro-vomeronasogram                                                  GSR = Galvanic Skin Response                                                    = Electrodermal Activity (EDA)                                              ST = Skin Temperature                                                       ______________________________________                                    

FIG. 9 depicts the electrophysiological effect of vomeropherins on thevomeronasal organ of 20 male subjects.

M=1,3,5(10)-Estratrien-3-ol

FIG. 10 depicts the synthesis of Estra-1,3,5(10),6-tetraen-3-ol andEstra-4,16-dien-3-ol.

FIG. 11 depicts the synthesis of compounds described in Examples 16through 19.

FIG. 12 illustrates the steps of cynthesis described in Examples 20through 24.

FIG. 13 illustrates the steps of synthesis described in Examples 25through 28.

FIG. 14 illustrates the steps of synthesis described in Examples 29through 30.

FIG. 15 illustrates the steps of synthesis described in Examples 31through 36.

FIG. 16 illustrates the steps of synthesis described in Examples 37through 39.

FIG. 17 illustrates the steps of synthesis described in Examples 40through 46.

FIG. 18 illustrates the steps of synthesis described in Examples 47through 49.

FIG. 19 illustrates the steps of synthesis described in Examples 50through 51.

FIGS. 20A, 20B and 20C illustrate the EVG, GSR and ST data on women,respectively, for 13 estranes on Chart 1.

FIGS. 21A, 21B and 21C illustrate the EVG, GSR and ST data on men,respectively, for 13 estranes on Chart 1.

FIGS. 22A and B through 34A and B illustrate the EEG data on men (A) andwomen (B) for the 13 estranes, respectively, identified in FIG. 20.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

An "affect" is a transient feeling state. Typical negative affects arefeelings of nervousness, tenseness, shame, anxiousness, irritability,anger, rage, and the like. "Moods" are longer lasting feeling statessuch as guilt, sadness, hopelessness, worthlessness, remorsefulness,misery, unhappiness and the like. "Character" traits are more permanentaspects of an individual's personality. Typical negative charactertraits are sensitivity, regretfulness, blameworthiness, stubbornness,resentfulness, bitterness, timidness, laziness and the like.

"Androstane steroids" are aliphatic polycyclic hydrocarbonscharacterized by a four-ring steroidal structure, with a methylation atthe 10- and 13- positions. An Androstene steroid is a subset ofAndrostanes, commonly understood to mean that the compound has at leastone double bond. Commonly, unless a compound is described as a gonane itis understood that the compound has an 18- carbon group. However, it isintended that 18-Nor-Androstaines are herein regarded as Androstanesteroids. Furthermore, all derivatives which have the structuralcharacteristics described above are also referred to generically hereinas Androstane steroids.

The following structure shows the four-ring teroidal structure common toAndrostene and Estrene steroids. In describing the location of groupsand substituents, the following numbering system will be employed:##STR2##

"Sexually dimorphic" refers to a difference in the effect of, orresponse to, a pharmaceutical agent between males and females of thesame species.

An "effective amount" of a drug is a range of quantity and/orconcentration which brings about a desired physiological and/orpsychological effect when administered to a subject in need of the drug.In the present case, a needy subject is one in need of hypothalamicmodulation or regulation, or a subject in need of alteration of aphysiological or behavioral characteristic normally affected by thehypothalamus. The effective amount of a given drug may vary dependingupon the route of administration. For example, when the steroid isadministered as a solution applied to the facial skin of a subject aneffective concentration is from about 1 to about 100 μg/ml, preferablyabout 10 to about 50 μg/mL and most preferably about 20 to about 30μg/ml. When the steroid is introduced directly into the VNO an effectiveamount is about 1 ρg to about 1 ng, more preferably about 10 ρg to about50 ρg. When the steroid is administered to the nasal passage byointment, cream, aerosol, or the like, an effective amount is about 100pg to about 100 micrograms, preferably about 1 ng to about 10micrograms. It follows that some drugs may be effective whenadministered by some routes, but not effective when administered byother routes.

The "hypothalamus" is the portion of the diencephalon comprising theventral wall of the third ventricle below the hypothalamic sulcus andincluding structures forming the ventricle floor, including the opticchiasma, tuber cinereum, infundibulum, and mammillary bodies. Thehypothalamus regulates the autonomic nervous system and controls severalphysiological and behavioral functions such as the so-called fight andflight responses, sexual motivation, water balance, sugar and fatmetabolism, hunger, regulation of body temperature, endocrinesecretions, and others. The hypothalamus is also the source ofvasopressin which regulates blood pressure, and oxytocin which inducesparturition and milk release. All hypothalamic functions are potentiallymodulatable by the semiochemical therapy described herein.

A "ligand", as used herein, is a molecule which acts as a chemicalsignal by specifically binding to a receptor molecule displayed on thesurface of a receptor cell, thereby initiating a signal transductionacross the cell surface. Binding of ligands to chemosensory receptorscan be measured. Chemosensory tissue, such as vomeronasalneuroepithelium or olfactory neuroepithelium, contains a multiplicity ofneuroreceptors cells, each displaying at least one cell surfacereceptor. Many of the receptor molecules have identical ligandspecificity. Therefore, when the tissue is exposed to a ligand for whichit has specificity (for example a exposure of the VNO to asemiochemical) a summated change in cell surface receptor potential canbe measured.

As used herein, "lower alkyl" means a branched or unbranched saturatedhydrocarbon chain of 1 to 4 carbons, such as, for example, methyl,ethyl, n-propyl, i-butyl and the like. "Alkoxy" as used herein is usedin its conventional sense to mean the group --OR where in R is an alkylas defined herein.

A "pheromone" is a substance that provides chemical means ofcommunication between members of the same species through secretion andnasus reception. In mammals pheromones are usually detected by receptorsin the vomeronasal organ of the nose. Commonly, pheromones effectdevelopment, reproduction and related behaviors. A "semiochemical" is amore general term which includes pheromones and describes a substancefrom any source which functions as a chemosensory messenger, binds to aspecific neuroepithelial receptor, and induces a physiological orbehavioral effect. A "vomeropherin" is a semiochemical whose physiologiceffect is mediated through the vomeronasal organ.

A picogram (pg) is equal to 0.001 nanograms (ng). A ng is equal to 0.001microgram (μg). A μg is equal to 0.001 mg.

II. Modes for Carrying Out the Invention

A. Estrenes Useful in the Invention

The invention is directed in part to certain Estrene steroids which arestructurally related to Estradiol (also referred to as1,3,5(10)-Etratriene-3,17β-diol). Steroids within the group arecharacterized by a aromatic 1,3,5(10) A-ring and a hydroxyl or hydroxylderivative at the 3-position.

Estrenes especially suitable for use in the present invention includethose where, independently, R₁ =oxo, β- hydroxy, hydrogen; R₂ =methyl,hydrogen; (as depicted in formula I).

Most of these steroids and their glucuronide, sulfate, cypionate, andbenzoate derivatives, are compounds known in the art and arecommercially available, e.g., from Sigma Chemical Co., Aldrich ChemicalCo., etc. Alkoxy derivatives and their synthesis are also known in theart and taught in U.S. Pat. No. 2,984,677, herein incorporated byreference.

Chart 1 includes estrenes to which the invention is directed, but do notlimit its scope. The synthesis diagrams that follow depict intermediateand substructure syntheses for the preparation of these estrenes:

    CHART 1       - ESTRANES       N       E 1 2 3 4       1      ##STR3##      ##STR4##      ##STR5##      ##STR6##     2      ##STR7##      ##STR8##      ##STR9##      ##STR10##     3      ##STR11##      ##STR12##      ##STR13##      ##STR14##     4      ##STR15##      ##STR16##      ##STR17##      ##STR18##     5      ##STR19##      ##STR20##      ##STR21##      ##STR22##     6      ##STR23##      ##STR24##      ##STR25##      ##STR26##     7      ##STR27##      ##STR28##      ##STR29##      ##STR30##     8      ##STR31##      ##STR32##      ##STR33##      ##STR34##     9      ##STR35##      ##STR36##      ##STR37##      ##STR38##     10      ##STR39##      ##STR40##      ##STR41##      ##STR42##     11      ##STR43##      ##STR44##      ##STR45##      ##STR46##     12      ##STR47##      ##STR48##      ##STR49##      ##STR50##

NOVEL ESTRANES

ESTRA-1,3,5(10),6,16-PENTAEN-3-YL ACETATE (Acetate of E4/N1)

ESTRA-1,3,5(10),7-TETRAEN-3-OL (E9/N2)

3-HYDROXY ESTRA-1,3,5(10),16-TETRAEN-6-ONE (E6/N1)

6-OXO ESTRA-1,3,5(10),16-TETRAEN-3-YL ACETATE (Acetate of E6/N1)

17-METHYLENE ESTRA-1,3,5,6,9-PENTAEN-3-OL (E10/N3)

ESTRA-1,3,5(10),16-TETRAEN-3,6β-DIOL (E12/N1)

6β-HYDROXYESTRA-1,3,5(10),16-TETRAEN-3-YL ACETATE (Acetate of E12/N1)

ESTRA-4,16-DIEN-3β-OL (E3/N1)17-METHYLENE-6-OXOESTRA-1,3,5(10)-TRIEN-3-YL ACETATE (Acetate of E6/N3)

ESTRA-4,9,16-TRIEN-3-ONE (E5/NI)

ESTRA-1,3,5,7,9,16-HEXAEN-3-OL (E10/N1)

ESTRA-1,3,5(10),6-TETRAEN-3-OL (E4/N2)

3-METHOXYESTRA-2,5(10),16-TRIENE (E8/N1)

10-HYDROXYESTRA-4,16-DIEN-3-ONE (E7/N1)

17-METHYLENEESTRA-1,3,5(10),7-TETRAEN-3-OL (E9/N3)

ESTRA-5(10),16-DIEN-3α-OL (3α epimer of E11/N1)

ESTRA-5(10),16-DIEN-3α-OL (E11/N1)

17-METHYLENEESTRA-4-EN-3-ONE (E1/N3)

ESTRA-1,3,5,7,9,16-HEXAEN-3-YL ACETATE (Acetate of E10/N1)

17-METHYLGONA-4,13(17)-DIEN-3β-OL (E3/N4)

ESTRA-1,3,5,(10),7,16-PENTAEN-3-OL (E9/N1)

3-METHOXY-17-METHYLENEESTRA-2,5(10)-DIEN (E8/N3)

17-METHYLENEESTRA-4-EN-3β-OL (E3/N3)

17-METHYLENEESTRA-1,3,5(10)-TRIENE-3,6β-DIOL (E12/N3)

ESTRA-1,3,5(10),7,16-PENTAEN-3-YL ACETATE (Acetate of E9/N1)

ESTRA-1,3,5(10),6,16-PENTAEN-3-OL (E4/N1)

17-METHYLENEESTRA-1,3,5(10),7-TETRAEN-3-YL ACETATE (Acetate of E9/N3)

SUBSTRUCTURE SYNTHESES

Referring to the preceding table, the following are exemplary synthesesfor intermediates in a given row (E1 through E12) or column (N1 throughN4). ##STR51## Synthesisable compounds therefore include these, togetherwith those derived from them; i.e., 17-Methyl-N1, 17β-Methyl-N2, or14α-Methyl-N4, in combination with E1, E2, E3, E5, E6, E7, E8, E11 orE12. ##STR52## Synthesisable compounds therefore include these, togetherwith those derived from them; i.e., 17-Fluoro-N1 in combination with El,E2, E3, E5, E6, E7, E11 or E12. In addition, 17-Iodo-N1 in combinationwith E2, E6 or E12.

B. Synthetic Methods

General procedures for synthetic reactions of steroids are known tothose skilled in art: (See for example, Fieser, L. F. and M. Fieser,Steroids, Reinhold, N.Y. 1959). Where time and temperature of reactionsmust be determined, these can be determined by a routine methodology.After addition of the required reagents, the mixture is stirred under aninert atmosphere and aliquots are removed at hourly intervals. Thealiquots are analyzed by means of thin-layer chromatography to check forthe disappearance of starting material, at which point the work-upprocedure is initiated. If the starting material is not consumed withintwenty-four hours, the mixture is heated to reflux and hourly aliquotsare analyzed, as before, until no starting material remains. In thiscase the mixture is allowed to cool before the work-up procedure isinitiated.

Alkoxy derivatives of Estrenes are prepared from their correspondinghydroxy steroids by reaction with an alkylating agent such astrimethyloxonium fluoroborate, triethyloxonium fluoroborate ormethylfluorosulfonate in an inert chlorocarbon solvent such as methylenechloride. Alternatively, alkylating agents such as alkyl halides, alkyltosylates, alkyl mesylates and dialkylsulfate may be used with a basesuch as silver oxide or barium oxide in polar, aprotic solvents as forexample, DMF, DMSO and hexamethylphosphoramide. Alternatively, a basesuch as K₂ CO₃ may be used in solvents such as ethanol or acetone.

Purification of the products is accomplished by means of chromatographyand/or crystallization, as known to those skilled in the art.

C. Pharmaceutical Compositions and Methods of Use

An embodiment of the subject invention is a method of altering thehypothalamic function of an individual. Another embodiment is alteringan autonomic function of an individual. These autonomic functionsinclude, but are not limited to, heart rate, respiratory rate, brainwave patterns (percentage alpha cortical activity), body temperature.Other embodiments include, but are not limited to, methods ofdiminishing negative affect, negative mood or negative character traitsof an individual. Another embodiment is a method of treating femalepremenstrual stress. All of these embodiments are accomplished by meansof the non-systemic, nasal administration of certain Estrene steroidsand combinations of Estrene steroids.

This particular mode of administration is distinguished from alternativemodes, such as ingestion or injection, in several important ways, theseby virtue of the direct contact with the VNO provided by the nasaladministration of the steroid ligand. In the methods of this invention,the appropriate ligand is administered directly to the chemoreceptors inthe nasal passage and the vomeronasal organ, without pills orneedles--i.e., noninvasively. Drug action is mediated through binding ofthe ligands, described herein, to specific receptors displayed byneuroepithelial cells in the nose, preferably in the VNO. This mode ofdrug action is through the nervous system and not through thecirculatory system--thus brain function can be affected withoutconsideration of the blood-brain barrier. These methods of treatmentprovide a direct means of affecting the hypothalamus through the nervoussystem because there is only one synaptic junction between pheromonereceptors and the hypothalamus. Because sensory nerves are addressed toa specific location in the brain, this method has a highly specific drugeffect, thereby greatly reducing the potential of undesirableside-effects.

VNO contact is important because the VNO is associated withchemoreceptive/pheromonal function. The VNO consists of a pair of blindtubular diverticula which are found at the inferior margin of the nasalseptum. The VNO contains neuro-epithelia, the axons of which have directsynapses to the amygdala and from there, to the hypothalamus. Theexistence of the VNO has been well documented in most terrestrialvertebrates including the human fetus; however, in adult humans it isgenerally thought to be rudimentary (See Johnson, et al., supra).

The active compounds described herein, or their sulfated, cypionated,benzoated, propionated, halogenated or glucuronated derivatives, may beadministered directly, but are preferably administered as compositions.They are prepared in a liquid dosage form such as, for example, liquids,suspensions or the like, preferably in unit dosage forms suitable forsingle administration of precise dosages. Liquid dosages may beadministered as nose drops or as an aerosol.

Alternatively, the active compound can be prepared as a creme or anointment composition and applied topically within the nasal cavity. Asanother alternative, delivery may occur by controlled release of theseagents by encapsulation either in bulk or at a microscopic level usingsynthetic polymers, such as silicone, and natural polymers such asgelatin and cellulose. The release rate can be controlled by properchoice of the polymeric system used to control the diffusion rate(Langer, R. S. and Peppas, N. A., Biomaterials 2,201, 1981). Naturalpolymers, such as gelatin and cellulose slowly dissolve in a matter ofminutes to hours while silicone remains intact for a period of months.The compositions will include a conventional pharmaceutical carrier orexcipient, one or more of the active Estrene compound(s) of Formula I.In addition, the compositions may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, etc.

The most likely means of communication of a putative human pheromone isthe inhalation of a naturally occurring pheromone present on the skin ofanother. Several 16-Androstene steroids, including5α-Androst-16-en-3α-ol and 5α-Androst-16-en-3-one,4,16-Androstadien-3-one, 5α-Androstadien-3β-ol, and perhaps5α-Androstadien-3α-ol, are naturally occurring in humans and may bepresent on the skin. It is estimated that the naturally occurringmaximum concentration of a 16-Androstene steroid on human skin is from 2to 7 ng/cm². During intimate contact it is estimated that a human wouldbe exposed to no more than 700 ng of a naturally occurring steroid.Since these compounds are relatively non-volatile, it is estimated that,even during intimate contact, a human subject would inhale no more than0.7 pg of a naturally occurring steroid from the skin of another. Fromthe amount inhaled only about 1% would reach the receptors of thevomeronasal organ. Thus the estimated maximum natural exposure tonaturally produced pheromones would be 0.007 pg.

The amount of active compound administered will of course, be dependenton the subject being treated, the severity of-the affliction, the mannerof administration, the frequency of administration, and the judgment ofthe prescribing physician. However, a single dosage of at least about 10picograms, delivered directly into the lumen of the vomeronasal organ,is effective in eliciting a transient autonomic response whenadministered to the nasal cavity, the dosage is about 100 picograms toabout 100 micrograms, preferably about 1 nanogram to about 10micrograms, more preferably about 10 nanograms to about 1 microgram. Thefrequency of administration is desirably in the range of an hourly doseto a monthly dose, preferably from 8 times/day to once every other day,more preferably 1 to 3 times per day. Ointments containing one or moreactive compounds and optional pharmaceutical adjuvants in a carrier,such as, for example, water, saline, aqueous dextrose, glycerol,ethanol, and the like, can be prepared using a base such as, forexample, petroleum jelly, lard, or lanolin.

Liquified pharmaceutically administrable compositions can, for example,be prepared by dissolving, dispersing, etc. an active compound asdefined above and optional pharmaceutical adjuvants in a carrier, suchas, for example, water, saline, aqueous dextrose, glycerol, ethanol, andthe like, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, etc. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, seeRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,15th Ed., 1975. The composition or formulation to be administered will,in any event, contain a quantity of one or more of the activecompound(s) in an amount effective to alleviate the symptoms of thesubject being treated.

For aerosol administration, the active ingredient is preferably suppliedin finely divided form along with a surfactant and a propellant. Typicalpercentages of active ingredients are 0.001 to 2% by weight, preferably0.004 to 0.10%.

Surfactants must, of course, be nontoxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, olestearic andoleic acids with an aliphatic polyhydric alcohol or its cyclic anhydridesuch as, for example, ethylene glycol, glycerol, erythritol, arabitol,mannitol, sorbitol, and hexitol anhydrides derived from sorbitol (thesorbitan esters sold under the trademark "Spans") and thepolyoxyethylene and polyoxypropylene derivatives of these esters. Mixedesters, such as mixed or natural glycerides, may be employed. Thepreferred surface-active agents are the oleates or sorbitan, e.g., thosesold under the trademarks "Arlacel C" (sorbitan sesquioleate), "Span 80"(sorbitan monooleate) and "Span 85" (sorbitan trioleate). The surfactantmay constitute 0.1-20% by weight of the composition, preferably 0.25-5%.

The balance of the composition is ordinarily propellant. Liquefiedpropellants are typically gases at ambient conditions, and are condensedunder pressure. Among suitable liquefied propellants are the loweralkanes containing up to five carbons, such as butane and propane;fluorinated or fluorochlorinated alkanes, such as are sold under thetrademark "Freon". Mixtures of the above may also be employed.

In producing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedactive ingredient and surfactant. The ingredients are thus maintained atan elevated pressure until released by action of the valve.

Yet another means of administration is topical application of a volatileliquid composition to the skin, preferably facial skin, of anindividual. The composition will usually contain an alcohol such asethanol or isopropanol. A pleasant odorant may also be included in thecomposition.

D. Measuring Affect. Mood and Character Traits

Feeling states associated with affects, moods and character traits aregenerally measured by use of a questionnaire. For example questionnairescomprising a number of adjectives which refer to feeling states may beadministered to an individual. The individual evaluates his or herfeeling state described by the adjective and rates the intensity of thefeeling on a numerical scale. Clustering of related adjectives andstatistical analysis of a subject's evaluation of each adjectiveprovides a basis for the measurement of various feeling states.

Alternatively, feeling states may be measured by autonomic changes, suchas those used in polygraphic evaluations (galvanic skin response, pulserate and the like). Cabanac, M. Annual Review of Physiology (1975)37:415; Hardy, J. D., "Body Temperature Regulation", Chapter 59, pp.1417. In: Medical Physiology. Vol. IIEd.: VB Mountcastle (1980); WolframBouscein. Electrodermal Activity (Plenum Press 1992). In addition,non-verbal cues such as facial expression and body posture may beevaluated.

III. Examples

The following examples are intended to illustrate but not to limit theinvention.

Abbreviations used in the examples are as follows: aq.=aqueous; RT.=roomtemperature; PE=petroleum ether (b.p. 50-70°); DMF=N,N-dimethylformamide; DMSO-dimethyl sulfoxide; THF=tetrahydrofuran.

Example 1--Synthesis of Estra-1,3,5(10,16-tetraen-3-ol (28)

The following method of synthesis is depicted in FIG. 1:

Estrone p-Toluenesulfonylhydrazone (27)

Estrone (26) (270 g, 1.00 mole) and p-toluenesulfonylhydrazide (232.8 g,1.25 mole) in dry methanol (2.5 liters) were heated under reflux for 20hours. The mixture was transferred to a conical flask and allowed tocool. The crystalline product was filtered off under suction and washedwith methanol (300 ml). Further crops of product were obtained bysequentially evaporating the filtrate to 2000 ml, 800 ml and 400 ml, andallowing to crystallize each time. Total yield was 433.5 g (99%) .

1,3,5(10),16-Estratetraen-3-ol (28)

Estrone p-toluenesulfonylhydrazone (27) (219.0 g, 500 m mole) in drytetrahydrofuran (8.0 liters) was cooled in a sodium chloride/ice bath.The mixture was mechanically stirred while n-butyl lithium (800 ml of a2.5M solution in hexane, 2.00 mole) was added via double-ended needle.The mixture was stirred at room temperature for 3 days. Ice (250 g) wasadded, followed by saturated ammonium chloride solution (500 ml). Thephases were mixed by stirring and then allowed to settle. The aqueousphase was removed via aspiration with teflon tube and extracted withether (500 ml). The two organic phases were sequentially washed with thesame batch of saturated sodium bicarbonate solution (500 ml) followed bysaturated sodium chloride solution (500 ml). The organic layers weredried (MgSO₄) and evaporated in vacuo to give crude product. This wassubjected to flash filtration on 500 g silica gel 60, 230-400 mesh,eluting with ethyl acetate/hexane (25:75, 2.5 liters) The filtrate wasevaporated in vacuo to give crystalline material. The product wasrecrystallized from methanol (300 ml)/water (75 ml) washing withmethanol (80 ml)/ water (20 ml). Further recrystallization from ethylacetate/hexane (12.5:87.5) gave pure product (88.9 g, 70%).

Example 2--Synthesis of Acyl derivatives of 1,3,5(10),16-Estratetraen-3-ol

To 1,3,5(10),16-Estratetraen-3-ol (254 mg, 1.00 mMole) in ether (10 ml)is added acetic anhydride (0.25 ml) (or propionic anhydride for thepropionate) followed by pyridine (0.25 ml) and the mixture is stirred atroom temperature for 16 hours. The mixture is poured into ice/water andextracted with ether (2×20 ml). The organic extracts are washed withwater, saturated copper sulfate solution, water, and saturated sodiumchloride solution, dried (MgSo₄) and evaporated in vacuo to give thecrude material. This is purified by flash chromatography on 17.5 gsilica gel 60 (230-400 mesh) eluting with 10%-12% ethyl acetate/hexaneto give the pure product (192 mg, 65%).

Example 3--Synthesis of Estra-4,16-dien-3-one (1)

To estra-1,3,5(10),16-tetraene-3-methyl ether (551.5 mg., 2.055 mmol) in8.6 ml of anhydrous THF, approximately 30 ml of anhydrous ammonia, and6.76 g of t-butyl alcohol was added lithium wire (0.24 g, 35 mg-atom)cut in small pieces. The reaction mixture was refluxed 4 1/2 h underargon, after which methanol (2.3 ml) was added and the ammonia wasallowed to boil off overnight. The residue was dissolved in 25 mL ofmethanol and was acidified to approximately pH 1 with 5N HCI. Afterheating in an oil bath between 55 and 70° C. for 15 min. the cooledhydrolysis mixture was partitioned between 25 ml of water and 50 ml ofethyl acetate and the aqueous phase was extracted with 25 ml of ethylacetate. The combined organic extracts were washed with 25 ml ofsaturated sodium bicarbonate and 25 mL of brine, dried over magnesiumsulfate, and filtered. Removal of solvent under reduced pressure yielded0.57 g of oily residue which was purified by flash chromatography onsilica gel (eluent: 15% ethyl acetate/hexane) followed byrecrystallization from pentane to give crystals (206.1 mg, 39%)homogeneous to TLC, m.p. 67-71° C.

Example 4--Synthesis of Estra-2,5(10),16-triene-3-methyl ether (2)

To Estra-1,3,5(10),16-tetraene-3-methyl ether (1.22 g, 4.54 mmole) in 19ml of anhydrous; THF, 14.99 g of t-butyl alcohol, and approximately 70ml of anhydrous ammonia was added lithium wire (0.53 g, 76 mg-atom) cutin small pieces. After refluxing under argon for 6 h the reaction wasquenched with 5 ml of methanol and ammolonia was allowed to boil offovernight. A suspension of the residue in 100 ml of water was extractedtwice with 100 ml portions of ethyl acetate and the combined organicextracts were washed with brine and dried over magnesium sulfate.Following solvent removal under reduced pressure the residue was flashchromatographed on silica gel using 1% ethyl acetate/hexane as eluentand then recrystallized from abs. ethanol to give fluffy white crystals(884.1 mg, 3.269 mmole, 72%), m.p. 72-73° C., homogeneous to TLC.

Example 5--Synthesis of Estra-5(10),16-dien-3-one (3)

Estra-2,5(10),16-triene-3-methyl ether (2) (646.3 mg, 2.390 mmole),dissolved in 50 ml of acetone was hydrolyzed for 6 h at room temperatureusing oxalic acid dihydrate (0.84 g, 6.7 mmole). The reaction mixturewas quenched with 25 ml of saturated sodium bicarbonate and thenextracted twice with 25 ml portions of ethyl acetate. The combinedorganic extracts were washed twice with 25 ml of brine, dried overmagnesium sulfate, filtered, and concentrated under reduced pressure.The residue was recrystallized from hexane to give product (462.5 mg,1.804 mmole, 75%), m.p. 112-116° C.

Example 6--Synthesis of Estra-5(10),16-dien-3-ols (4)

Estra-5(10),16-dien-3-one (3) (301.1 mg, 1.174 mmole), in 6 mL ofanhydrous ether was reduced for 1 h at room temperature using lithiumaluminum hydride (50.0 mg, 1.32 mmole). After quenching with sodiumsulfate decahydrate (2.00 g) for 10 min. the suspension was filteredthrough Celite and the residue washed with four 25 mL portions of ether.The combined filtrates were concentrated under reduced pressure andpurified by flash chromatography (silica gel, 5% ethyl acetate/ hexaneseluent) with subsequent preparative TLC of mixed fractions. The morepolar product could be recrystallized with considerable loss fromaqueous ethanol to give 4.8 mg of solid. The less polar product wasrecrystallized from aqueous methanol to give white crystals (59.5 mg),m.p. 98-100° C. Total yield was 64.3 mg (0.249 mmol, 21%).

Example 7--Synthesis of Estra-4,9,16-trien-3-one (5)

Estra-5(10),16-dien-3-one (3) (0.38 g, 1.5 mmole), in pyridine (5.0 mL,62 mmol) was cooled in an ice-salt bath to -13° C. and pyridiniumbromide perbromide (1.58 g, 4.94 mmole) was added in small portions sothat T<-4° C. After swirling 1 min. phenol (0.25 g, 2.7 mmole) was addedand reaction continued 24 h at room temperature. Ethyl acetate (50 ml)was added and the reaction mixture was washed with 25 ml of 1N HCI, two25 ml portions of saturated copper sulfate, 25 ml of 5% sodiumhydroxide, and 25 ml of brine. After drying over magnesium sulfate,filtration, and concentration under reduced pressure the residue wastaken up in 10 mL of abs. ethanol, granular zinc (0.33 g, 5.0 mg-atom)was added, and the mixture was refluxed 1/2 h. The supernatant wasremoved, the residue was washed with 10 mL of abs. ethanol, and thecombined supernatants were concentrated under reduced pressure. Theresulting resin was flash chromatographed on silica gel using 15% ethylacetate/ hexane as eluent. Appropriate fractions were pooled,concentrated, and then recrystallized from hexane to give solid product(117.5 mg, 0.4619 mmol, 31%), m.p. 87-92° C.

Example 8--Synthesis of Estra-1,3,5(10,16-tetraen-6-one-3-acetate (6)

Chromium trioxide (13.40 g, 0.1340 mol) was suspended in 200 mL ofmethylene chloride and then cooled to -10° C. in an ice-salt bath.3,5-Dimethylpyrazole (12.90 g, 0.1342 mol) was added and the mixture wasstirred 20 min. Estra-1,3,5(10),16-tetraen-3-yl acetate (4.00 g, 13.5mmol) in a chilled solution of 20 mL of methylene chloride was added andthe reaction stirred 2 h, during which time T<-8° C. The mixture wasthen filtered through 200 g of silica gel and the product was elutedwith further methylene chloride. After combining and concentratingappropriate fractions the crude product was flash chromatographed onsilica gel using 15% ethyl acetate/hexane as eluent. Pooling ofappropriate fractions and concentration under reduced pressure yielded awhite solid (0.92 g, 3.0 mmol, 22%), m.p. 87-103° C.

Example 9--Synthesis of Estra-1,3,5(10),16-tetraen-3-ol-6-one (7)

Estra-1,3,5(10),16-tetraen-6-one-3-acetate (203.1 mg, 0.6543 mmol) in 30of methanol was saponified with 1.5 mL of 5% (w/w) sodium hydroxide for40 min. The reaction mixture was concentrated under reduced pressure,taken up in 50 mL of water, neutralized with 1 N HCI, and extractedthree times with 25 mL portions of methylene chloride. The combinedorganic extracts were washed with 50 mL of brine, dried over magnesiumsulfate, filtered, and concentrated to give a white solid which waspurified by recrystallization from benzene/hexane and preparative TLC togive white crystalline solid (52.8 mg, 0.197 mmol, 30%), m.p. 188-191°C.

Example 10--Synthesis of Estra-1,3,5(10),16-tetraen-6α-ol-3-yl acetate(8)

Estra-1,3,5(10),16-tetraen-6-one-3-yl-acetate (6) (421.4 mg, 1.358mmol), suspended in 35 mL of 95% ethanol was reduced with sodiumborohydride (98.8 mg, 2.61 mmol) for 100 min. at room temperature. Afterconcentrating under reduced pressure the residue was suspended in 25 mLof water, neutralized with 1N HCl, and extracted three times with 25 mLportions of methylene chloride. The combined organic extracts werewashed with 25 mL of brine, dried over magnesium sulfate, filtered, andconcentrated. The resulting white foam was flash chromatographed onsilica gel using 25% ethyl acetate/hexane as eluent. Combining fractionsand concentration gave a white solid (0.12 g, 0.38 mmol, 28%), m.p.209-212° C.

Example 11--Synthesis of Estra-1,3,5(10),16-tetraene-3,6-diol (9)

To a suspension of lithium aluminum hydride (LAH, 95%, 46.9 mg, 1.17mmol) in 5 mL of anhydrous THF was addedestra-1,3,5(10),16-tetraen-6-one-3-yl-acetate (6) (422.9 mg, 1.360 mmol)in 5 mL of anhydrous THF dropwise, with stirring. The reaction wasstirred 50 min., after which further LAH (46.5 mg, 1.16 mmol) was addedand the reaction stirred 22 h. After refluxing 4 h TLC still showedstarting material. The reaction was quenched with 0.5 mL of water +0.5mL of 20% (w/w) sulfuric acid and concentrated under reduced pressure.The residue was extracted four times with 10 mL portions of hot ethylacetate and filtered through Celite. The combined filtrates wereconcentrated and purified twice by flash chromatography to give solidproduct. (0.05 g, 0.2 mmol, 10%), m.p. 150-157° C.

Example 12--Synthesis of Estra-1,3,5(10),7-tetraen-3-ol (10)

To a suspension of equilin (100.2 mg, 0.3733 mmol) in 2 mL of diethyleneglycol were added hydrazine (59 μL, 1.9 mmol) and potassium hydroxide(0.04 g, 0.7 mmol). The mixture was stirred in an oil bath at 200-214°C. for 2 h, after which the cooled reaction was diluted with 10 mL ofwater, neutralized with 1N HCl, and extracted three times with 25 mL ofether. The combined organic extracts were washed with 10 mL of brine,dried over magnesium sulfate, filtered, concentrated, and purified bypreparative TLC (silica gel, 15% ethyl acetate/hexane eluent) to give ayellow resin. Product was further purified by decolorizing with charcoaland recrystallization from aqueous ethanol to give tan crystals (13.2mg, 51.9 μM, 14%), m.p. 130-134° C.

Example 13--Synthesis of 20-Homoestra-1,3,5(10), 6,8,17-hexaen-3-ol (11)

A suspension of triphenylmethylphosphonium bromide (671.0 mg, 1.878 mg)and potassium t-butoxide (212.1 mg, 1.890 mmol) in 2.1 mL of anhydrousDMSO was heated in a 76-86° C. bath under argon for 1 h, after whichequilenin (100.1 mg, 0.3579 mmol) in 2.1 mL of anh. DMSO was added andthe green solution was stirred 1 h. After cooling 10 mL of ice-1N HClwere added and the mixture was extracted with three 10 mL portions ofether. The combined organic extracts were washed with 10 mL of saturatedsodium bicarbonate +10 mL of brine, dried over magnesium sulfate,filtered through Celite, and concentrated under reduced pressure. Theresidual orange oil was purified by preparative TLC (silica gel, 25%ethyl acetate/hexane) to give product (75.5 mg, 0.286 mmol, 76%)homogeneous to TLC, m.p. 113-121° C.

Example 14--Synthesis of Estra-1,3,5(10),6-tetraen-3-ol (17)

Estra-1,3,5(10),6-tetraen-3-ol-17-one (91.1 mg, 0.339 mmol), hydrazine(54 μL, 1.7 mmol), and potassium hydroxide (0.06 g) in 1.8 mL ofdiethylene glycol were heated in a 200° C. bath under argon for 2 h.After cooling to RT 10 mL of water were added and the solution wasacidified to pH≈2 with 1N HCl. The resulting suspension was extractedthree times with 10 mL of ether and the combined organic extracts werewashed with 10 mL of brine, dried over magnesium sulfate, filteredthrough Celite, and concentrated under reduced pressure. The crude solidwas purified by preparative TLC (25% ethyl acetate/hexane on silica gel)to give product homogeneous to TLC (5.9 mg, 23 μmol, 7%).

Example 15--Synthesis of Estra-4,16-dien-3-ol (18)

To estra-4,16-dien-3-one, (1) (87.2 mg, 0.340 mmol) in 1.7 mL of anh.ether was added lithium aluminum hydride (15.0 mg, 0.395 mmol) and thesuspension was stirred 17 min. Reaction was then agitated 10 min. with0.50 g of sodium sulfate decahydrate and filtered through Celite. Theresidue was washed with three 10 mL portions of ether and the combinedfiltrates were concentrated under reduced pressure. Preparative TLC (5%ethyl acetate/dichloro-methane on silica gel) gave crude product (50.0mg) as a yellow resin. This could be rechromatographed untilsufficiently pure.

Example 16--Estra-4,16-dien-3-one (9)

This synthesis is depicted in FIG. 11. 19-Nor-testosterone (XIX) iscommercially available, e.g., from Chemical Dynamics Corp. It providesthe starting material for 19-Nor-16-androstene derivatives.19-Nor-testosterone (XIX) was converted into the acetate (Hartman, J. A.et al., J. Am. Chem. Soc. (1956) 78:5662) with acetanhydride andpyridine. (a) A solution of this acetate (4.8 g, 15.17 mmol) in toluene(10 ml) was pyrolyzed (b) at 540° (200 Torr, slow N₂ -stream) in a glasstube packed with quartz pieces. Chromatography of the crude pyrolysate(3.1 g) on silica gel (150 g) with CH₂ Cl₂ gave 1.1 g (28%) of thehomogenous oily ketone 9; +57.9° (C 1) ((27): m.p. 71-73°). -IR.(CHCl₃): 1660s, 1615m, 1585w, ¹ H-NMR. (90 MHz): 0.84 (s, 3 H); 5.82 (m,2 H); 5.87 (br. s, 1 H).

Example 17--Estr-16-en-3-one (10)

This synthesis is depicted in FIG. 11. 19-nor-testosterone was reducedto 19-nor-5a-Androstan-17 -ol-3 -one (XX) with lithium and ammonia (c)according to the method of Villotti, R., et al. (J. Am. Chem. Soc.(1960) 82:5693). Androsta-!5a,17-diol-3-one (XX) was converted into theacetate (Hartman, J. A. et al., J. Am. Chem. Soc. (1956) 78:5662) withacetanhydride and pyridine (a). A solution of17B-acetoxy-5a-Estran-3-one (8.0 g, 25.1 mmol) in octane/acetone 10:1(22 ml) was pyrolyzed (b) at 550° (200 Torr, slow N₂ -stream)Chromatography of the crude product (5.4 g) on silica gel (600 g) withCH₂ Cl₂ and recrystallization of the homogenous fractions from PE gave3.13 g (48.3%). of the pure ketone 10. M.p. 51-54°, [a]- +72.80 (C 1.0).-IR. (CHCl₃): 1705s, 1585w, - ¹ H-NMR. (90 MHz): C0.79 (s, 3 H); 5.71(m, 1 H); 5.87 (m, 1 H).

Example 18--Estra-16-en-3α-ol (11)

This synthesis is depicted in FIG. 11. L-Selectride (d, lithiumtri(sec-butyl)hydridoborate, 4 ml of a 1M solution in THF, 4 mmol) wasadded dropwise at 0° to a solution of ketone 10 (800 mg, 3.10 mmol) indry ether (5 ml). After stirring for 1 h at 0°, water was added (10 ml).The boranes were oxidized by adding 10% aq. NaOH-solution (5 ml),followed by 30% aq. H₂ O₂ -solution (3 ml) and stirring for 3 h at RT.After workup (ether), the crude product (790 mg, Ca. 9:1 mixture of 11and 12) was chromatographed on silica gel with CH₂ Cl₂ to give 700 mg(87%) of pure alcohol 11. M.p. 119-120°→123-124° (from PE), [a]_(D)+40.6° (C=1.0). - IR. (CHCl₃): 3640m, 3500 br., 1585w. - ¹ H-NMR. (90MHz): 0.78 (s, 3 H); 4.09 (m, w_(1/2) ≈8, 1 H); 5.71 (m, 1 H), 5.87 (m,1 H).

Example 19--Estra-16-en-3B-ol (12)

This synthesis is depicted in FIG. 11. A solution of the ketone 10 (800mg, 3.10 mmol) in dry ether (5 ml) was added dropwise at RT. to a slurryof LiAlH₄ (38 mg, 1 mmol) in ether (3 ml) (e). After 1 h, the mixturewas hydrolyzed with 10% aq. H₂ SO₄. After workup (ether), the crudeproduct (802 mg, 9:1-mixture of 12 and 11) was chromatographed on silicagel with CH₂ Cl₂. A small fraction of 11 (70 mg) was eluted first,followed by the main fraction of 12 (705 mg, 87%). M.p. 113-115°, [a]-+36.3° (C=1.0). -IR. (CHCl₃): 3640 m, 3500 br., 1585 w. - ¹ H-NMR. (90MHz): 0.78 (s, 3 H); 3.60 (m, w_(1/2) ≈ (m, 20, 1 H); 5.71 (m, 1 H),5.87 (m, 1 H).

Example 20--Alternative Synthesis of Estra-4,16-dien-3-one 1

Estra4,16-dien-3-one, 1: To estra-1,3,5(10),16-tetraene-3-methyl ether(551.5 mg, 2.055 mmol) in 8.6 mL of anhydrous THF, approximately 30 mLof anhydrous ammonia, and 6.76 g of t-butyl alcohol was added lithiumwire (0.24 g, 35 mg-atom) cut in small pieces. The reaction mixture wasrefluxed 4 1/2 h under argon, after which methanol (2.3 mL) was addedand the ammolonia was allowed to boil off overnight. The residue wasdissolved in 25 mL of methanol and was acidified to approximately pH 1with 5N HCl. After heating in an oil bath between 55 and 70° C. for 15min. the cooled hydrolysis mixture was partitioned between 25 mL ofwater and 50 mL of ethyl acetate and the aqueous phase was extractedwith 25 mL of ethyl acetate. The combined organic extracts were washedwith 25 mL of saturated sodium bicarbonate and 25 mL of brine, driedover magnesium sulfate, and filtered. Removal of solvent under reducedpressure yielded 0.57 g of oily residue which was purified by flashchromatography on silica gel (eluent: 15% ethyl acetate/hexane) followedby recrystallization from pentane to give crystals (206.1 mg, 39%homogeneous to TLC, m.p. 67-71° C. (NA-1993A-38, 42D)

Example 21--Estra-2,5(10),16-triene-3-methyl ether, 2

To estra-1,3,5(10),16-tetraene-3-methyl ether (1.22 g, 4.54 mmol) in 19mL of anhydrous THF, 14.99 g of t-butyl alcohol, and approximately 70 mLof anhydrous ammonia was added lithium wire (0.53 g, 76 mg-atom) cut insmall pieces. See FIG. 12. After refluxing under argon for 6 h thereaction was quenched with 5 mL of methanol and ammolonia was allowed toboil off overnight. A suspension of the residue in 100 mL of water wasextracted twice with 100 mL portions of ethyl acetate and the combinedorganic extracts were washed with brine and dried over magnesiumsulfate. Following solvent removal under reduced pressure the residuewas flash chromatographed on silica gel using 1% ethyl acetate/hexane aseluent and then recrystallized from abs. ethanol to give fluffy whitecrystals (884.1 mg, 3.269 mmol, 72%), m.p. 72-73° C., homogeneous toTLC. (NA-1993A-74, 77A)

Example 22--Estra-5(10),16-dien-3-one, 3

Estra-2,5(10),16-triene-3-methyl ether, 2 (646.3 mg, 2.390 mmol),dissolved in 50 mL of acetone was hydrolyzed for 6 h at room temperatureusing oxalic acid dihydrate (0.84 g, 6.7 mmol). See FIG. 12. Thereaction mixture was quenched with 25 mL of saturated sodium bicarbonateand then extracted twice with 25 mL portions of ethyl acetate. Thecombined organic extracts were washed twice with 25 mL of brine, driedover magnesium sulfate, filtered, and concentrated under reducedpressure. The residue was recrystallized from hexane to give product(462.5 mg, 1.804 mmol, 75%), m.p. 112-116° C. (NA- 1993A-78A)

Example 23--Estra-5(10),16-dien-3-ols 4

Estra-5(10),16-dien-3-one, 3 (301.1 mg, 1.174 mmol), in 6 mL ofanhydrous ether was reduced for 1 h at room temperature using lithiumaluminum hydride (50.0 mg, 1.32 mmol). See FIG. 12. After quenching withsodium sulfate decahydrate (2.00 g) for 10 min. the suspension wasfiltered through Celite and the residue washed with four 25 mL portionsof ether. The combined filtrates were concentrated under reducedpressure and purified by flash chromatography (silica gel, 5% ethylacetate/hexanes eluent) with subsequent preparative TLC of mixedfractions. The more polar product could be recrystallized withconsiderable loss from aqueous ethanol to give 4.8 mg of solid. The lesspolar product was recrystallized from aqueous methanol to give whitecrystals (59.5 mg), m.p. 98-100° C. Total yield was 64.3 mg (0.249 mmol,21%). (NA-1993A-80, 83A, 85A)

Example 24--Estra-4,9,16-trien-3-one 5

Estra-5(10),16-dien-3-one, 3 (0.38 g, 1.5 mmol), in pyridine (5.0 mL, 62mmol) was cooled in an ice-salt bath to -13° C. and pyridinium bromideperbromide (1.58 g, 4.94 mmol) was added in small portions so that T<"4°C. After swirling 1 min. phenol (0.25 g, 2.7 mmol) was added andreaction continued 24 h at room temperature. See FIG. 12. Ethyl acetate(50 mL) was added and the reaction mixture was washed with 25 mL of 1NHCl, two 25 mL portions of saturated copper sulfate, 25 mL of 5% sodiumhydroxide, and 25 mL of brine. After drying over magnesium sulfate,filtration, and concentration under reduced pressure the residue wastaken up in 10 mL of abs. ethanol, granular zinc (0.33 g, 5.0 mg-atom)was added, and the mixture was refluxed 1/2 h. The supernatant wasremoved, the residue was washed with 10 mL of abs. ethanol, and thecombined supernatants were concentrated under reduced pressure. Theresulting resin was flash chromatographed on silica gel using 15% ethylacetate/hexane as eluent. Appropriate fractions were pooled,concentrated, and then recrystallized from hexane to give solid product(117.5 mg, 0.4619 mmol, 31%), m.p. 87-92° C. (NA-1993A-62, 65B)

Example 25--Estra-1,3,5(10),16-tetraen-6-one-3-acetate 6

Chromium trioxide (13.40 g, 0.1340 mol) was suspended in 200 mL ofmethylene chloride and then cooled to -10° C. in an ice-salt bath.3,5-Dimethylpyrazole (12.90 g, 0.1342 mol) was added and the mixture wasstirred 20 min. See FIG. 13. Estra-1,3,5(10),16-tetraen-3-yl acetate(4.00 g, 13.5 mmol) in a chilled solution of 20 mL of methylene chloridewas added and the reaction stirred 2 h, during which time T<-8° C. Themixture was then filtered through 200 g of silica gel and the productwas eluted with further methylene chloride. After combining andconcentrating appropriate fractions the crude product was flashchromatographed on silica gel using 15% ethyl acetate/hexane as eluent.Pooling of appropriate fractions and concentration under reducedpressure yielded a white solid (0.92 g, 3.0 mmol, 22%), m.p. 87-103° C.(NA- 1993B-39B)

Example 26--Estra-1,3,5(10),16-tetraen-3-ol-6-one, 7

Estra-1,3,5(10),16-tetraen-6-one-3-acetate (203.1 mg, 0.6543 mmol) in 30of methanol was saponified with 1.5 mL of 5% (w/w) sodium hydroxide for40 min. See FIG. 13. The reaction mixture was concentrated under reducedpressure, taken up in 50 mL of water, neutralized with 1N HCl, andextracted three times with 25 mL portions of methylene chloride. Thecombined organic extracts were washed with 50 mL of brine, dried overmagnesium sulfate, filtered, and concentrated to give a white solidwhich was purified by recrystallization from benzene/hexane andpreparative TLC to give white crystalline solid (52.8 mg, 0.197 mmol,30%), m.p. 188-191° C. (NA-1993B-24, 27B)

Example 27--Estra-1,3,5(10),16-tetraen-6α-ol-3-yl acetate, 8

Estra-1,3,5(10),16-tetraen-6-one-3-yl acetate, 6 (421.4 mg, 1.358 mmol),suspended in 35 mL of 95% ethanol was reduced with sodium borohydride(98.8 mg, 2.61 mmol) for 100 min. at room temperature. See FIG. 13.After concentrating under reduced pressure the residue was suspended in25 mL of water, neutralized with 1N HCl, and extracted three times with25 mL portions of methylene chloride. The combined organic extracts werewashed with 25 mL of brine, dried over magnesium sulfate, filtered, andconcentrated. The resulting white foam was flash chromatographed onsilica gel using 25% ethyl acetate/hexane as eluent. Combining fractionsand concentration gave a white solid (0.12 g, 0.38 mmol, 28%), m.p.209-212° C. (NA- 1993B-42D)

Example 28--Estra-1,3,5(10),16-tetraene-3,6-diol, 9

To a suspension of lithium aluminum hydride (LAH, 95%, 46.9 mg, 1.17mmol) in 5 mL of anhydrous THF was addedestra-1,3,5(10),16-tetraen-6-one-3-yl acetate, 6 (422.9 mg, 1.360 mmol)in 5 mL of anhydrous THF dropwise, with stirring. See FIG. 13. Thereaction was stirred 50 min., after which further LAH (46.5 mg, 1.16mmol) was added and the reaction stirred 22 h. After refluxing 4 h TLCstill showed starting material. The reaction was quenched with 0.5 mL ofwater +0.5 mL of 20% (w/w) sulfuric acid and concentrated under reducedpressure. The residue was extracted four times with 10 mL portions ofhot ethyl acetate and filtered through Celite. The combined filtrateswere concentrated and purified twice by flash chromatography to givesolid product (0.05 g, 0.2 mmol, 10%), m.p. 150-157° C. (NA-1993B-29,32B)

Example 29--Estra-1,3,5(10),7-tetraen-3-ol, 10

To a suspension of equilin (100.2 mg, 0.3733 mmol) in 2 mL of diethyleneglycol were added hydrazine (59 μL, 1.9 mmol) and potassium hydroxide(0.04 g, 0.7 mmol). See FIG. 14. The mixture was stirred in an oil bathat 200-214° C for 2 h, after which the cooled reaction was diluted with10 mL of water, neutralized with 1N HCl, and extracted three times with25 mL of ether. The combined organic extracts were washed with 10 mL ofbrine, dried over magnesium sulfate, filtered, concentrated, andpurified by preparative TLC (silica gel, 15% ethyl acetate/hexaneeluent) to give a yellow resin. Product was further purified bydecolorizing with charcoal and recrystallization from aqueous ethanol togive tan crystals (13.2 mg, 51.9 μM, 14%), m.p. 130-134° C.(NA-1993B-25C)

Example 30--20-Homoestra-1,3,5(10),6,8,17-hexaen-3-ol, 11

A suspension of triphenylmethylphosphonium bromide (671.0 mg, 1.878 mg)and potassium t-butoxide (212.1 mg, 1.890 mmol) in 2.1 mL of anhydrousDMSO was heated in a 76-86° C. bath under argon for 1 h, after whichequilenin (100.1 mg, 0.3579 mmol) in 2.1 mL of anh. See FIG. 14. DMSOwas added and the green solution was stirred 1 h. After cooling 10 mL ofice-1N HCl were added and the mixture was extracted with three 10 mLportions of ether. The combined organic extracts were washed with 10 mLof saturated sodium bicarbonate +10 mL of brine, dried over magnesiumsulfate, filtered through celite, concentrated and purified.

Example 31--Estra-1,3,5(10),6-tetraen-3-ol-17-(p-toluenesulfonyl)hydrazone 1

A suspension of 6-dehydroestrone (538.0 mg, 2.004 mmol) andp-toluenesulfonylhydrazide (p-TsNHNH₂, 466.6 mg, 2.506 mmol) in anh.methanol (5.4 mL) was refluxed 25 h with exclusion of moisture. See FIG.15. After concentrating under reduced pressure the reaction residue wasflash chromatographed (50% ethyl acetate/hexanes on silica gel) to givean off-white foam (942.5 mg), representing >100% yield. (NA-1994A-295A)

Example 32--Estra-1,3,5(10),6,16-pentaen-3-ol, 2

To a cooled (ice water bath) solution of crude estral,3,5(10),6-tetraen-3-ol-17-(p-toluenesulfonyl)hydrazone (1, 942.5 mg,≦2.004 mmol) in tetrahydrofuran (THF) under argon was addedn-butyllithium (2.5M in hexane, 3.2 mL, 8.0 mmol) dropwise withstirring, over a period of 7 min. See FIG. 15. Stirring was continued 48h, during which the reaction was allowed to gradually warm to roomtemperature. 50 mL of 1N hydrochloric acid were added and the reactionmixture was extracted with three 25 mL portions of ether. The combinedorganic extracts were washed with 50 mL of brine, dried over magnesiumsulfate, and filtered through diatomaceous earth. The residue was washedwith 10 mL of ether and the combined filtrates were concentrated underreduced pressure. Crude product was purified by flash chromatography(20% ethyl acetate/hexanes on silica gel) and preparative TLC (20% ethylacetate/hexanes on silica gel GF, 1000μ thickness) to give a white,crystalline film (134.5 mg, 0.5331 mmol, 27%) homogeneous to TLC (20%ethyl acetate/hexanes on silica gel, R_(f) 0.39). (NA-1994A-307A)

Example 33--Estra-1,3,5(10),6, 16-pentaen-3-yl acetate, 3

A solution of estra-1,3,5(10),16-pentaen-3-ol (2, 97.9 mg, 0388 mmol) inanh. pyridine (1.3 mL, 16 mmol) and acetic anhydride (0.18 mL, 1.9 mmol)was stirred 24 h, after which ethyl acetate (15 mL) was added and themixture washed with three 5 mL aliquots of 1N hydrochloric acid +5 mL ofsaturated sodium bicarbonate +5 mL brine, dried over magnesium sulfate,and filtered through diatomaceous earth. See FIG. 15. The residue waswashed with 5 mL of ethyl acetate and the combined filtrates wereconcentrated under reduced pressure. Preparative TLC (10% ethylacetate/hexanes on silica gel GF, 1000μ thickness) of the residue gave aslightly yellow crystalline solid (74.9 mg, 0.254 mmol, 66%) homogeneousto TLC (10% ethyl acetate/hexanes on silica gel, R_(f) 0.40). (NA-1994B-21B)

Example 34--Estra-1,3,5(10),7-tetraen-3-ol-17-(p-toluenesulfonyl)hydrazone, 4

Equilin (500.1 mg, 1.863 mmol) and p-TsNHNH₂ (433.7 mg, 2.329 mmol)suspended in anh. methanol (5.0 mL) were refluxed 24 h with exclusion ofmoisture. See FIG. 15. After concentrating under reduced pressure theresidual reaction mixture was flash chromatographed (35% ethylacetate/hexanes on silica gel) to give a white foam (899.9 mg)representing >100% yield. (NA-1994A-246B)

Example 35--Estra-1,3,5(10),7,16-pentaen-3-ol, 5

To a cooled (ice water bath) solution of crudeestra-1,3,5(10),7-tetraen-3-ol,-17-(p-toluenesulfonyl) hydrazone (4,899.9 mg, <1.363 mmol) in anh. THF (20 mL) under argon was addedn-butyllithium (2.5M in hexane, 3.0 mL, 7.5 mmol) dropwise with stirringover a period of 3 min. See FIG. 15. Stirring was continued 48 h, duringwhich the reaction was allowed to gradually warm to room temperature.The reaction was poured into 50 mL of 1N hydrochloric acid and themixture was extracted with three 25 mL portions of ether. The combinedorganic extracts were washed with 50 mL of brine, dried over magnesiumsulfate, and filtered through diatomaceous earth. The residue was washedwith 10 mL of ether and the combined filtrates were concentrated underreduced pressure. The product was flashed chromatographed (20% ethylacetate/hexanes on silica gel) and decolorized with carbon to give ayellow crystalline solid (274.8 mg, 1.089 mmol, 58%). (NA-1994A-278A)

Example 36--Estra-1,3,5(10),7,16-pentaen-3-yl acetate, 6

A solution of estra-1,3,5(10),7,16-pentaen-3-ol (5, 192.1 mg, 0.7612mmol) in anh. pyridine (2.6 mL, 32 mmol) and acetic anhydride (0.36 mL,3.8 mmol) was stirred 6 h, after which 30 mL of ethyl acetate wereadded. The mixture was washed with three 10 mL portions of 1Nhydrochloric acid +10 mL of saturated sodium bicarbonate +10 mL ofbrine, dried over magnesium sulfate, and filtered through diatomaceousearth. See Example 15. The residue was washed with 10 mL of ethylacetate and the combined filtrates were concentrated under reducedpressure. Preparative TLC (5% ethyl acetate/hexanes on silica gel GF,1000μ thickness) and recrystallization from aqueous ethanol gave finewhite needles (78.6 mg, 0.267 mmol, 35%), m.p. 77-80° C. TLC (4% ethylacetate/hexanes on silica gel) showed 2 spots; at R_(f) 0.21 and 0.24.(NA-1994A-286A)

Example 37--Estra-1,3,5(10),6,8-pentaen-3-ol-17-(p-toluenesulfonylhydrazone, 7

Equilenin (0.6559 mg, 2.463 mmol) and p-TsNHNH₂ (573.6 mg, 3.080mmol)suspended in anh. methanol (8.2 mL) were refluxed 24 h with exclusion ofmoisture. See FIG. 16. After cooling and concentrating under reducedpressure the reaction mixture was flash chromatographed (35-40% ethylacetate/hexanes 57%), m.p. 95-96° C. TLC (2% ethyl acetate/hexanes onsilica gel) showed product (R_(f) 0.1) contained a trace contaminant atthe origin. (NA-1994A-273B)

Example 38--Estra-1,3,5(10),6,8,16-hexaen-3-oI, 8

To a cooled (ice water bath) solution of crudeestra-1,3,5(10)6,8-pentaen-3-ol 17-(p-toluenesulfonyl)hydrozne (7,1.0887 g, <2463 mmol) in anh. THF (25 mL) under argon was addedn-butyllithium (2.5M in hexane, 3.9 mL, 9.8 mmol) dropwise with stirringover 2 min. See FIG. 16. Stirring was continued 3 days, during which thereaction was allowed to gradually warm to room temperature. 50 mL of 1Nhydrochloric acid-ice were added and the mixture was extracted threetimes with 25 mL portions of ether. The combined organic extracts werewashed with 50 mL of brine, dried over magnesium sulfate, and filteredthrough diatomaceous earth. The residue was washed 10 mL of ether andthe combined filtrates were concentrated under reduced pressure. Flashchromatography (20% ethyl acetate/hexanes on silica gel) andrecrystallization from aqueous ethanol with charcoal decolorization gavetan platelets (245.8 mg, 0.9819 mmol, 40%), m.p. 162-163° C.(NA-1994A-269A)

Example 39--Estra-1,3,5(10),6,8,16-hexaen-3-yl acetate, 9

A solution of estra-1,3,5(10),6,8,16-hexaen-3-ol Q8, 148.8 mg, 0.5944mmol) in anh. pyridine (2.0 mL, 25 mmol) and acetic anhydride (0.28 mL,3.0 mmol) was stirred 6 h, after which ethyl acetate (20 mL) was added.See FIG. 16. The mixture was washed with three 10 mL portions of 1Nhydrochloric acid +10 mL of saturated sodium bicarbonate +10 mL ofbrine, dried over sodium sulfate, and filtered. The residue was washedwith 5 mL of ethyl acetate and the combined filtrates were concentratedunder reduced pressure. Recrystallization from 95% ethanol gave lustrouswhite platelets (99.4 mg, 0.340 mmol, 55%, m.p. 95-96° C. TLC (2% ethylacetate/hazanes on silica gel) showed product (R_(f) 0.1) contained atrace contaminant at the origin.

Example 40--17-Methylenestra-1,3,5(10)-trien-3-ol, 10

A suspension of methyltriphenylphosphonium bromide (100.03 g, 0.28001mol) and potassium t-butoxide (31.42 g, 0.2800 mol) in anh.dimethylsulfoxide (DMSO, 320 mL) under argon was stirred in an oil bath(68-81° C.) 1 h, after which estrone (15.14 g, 55.99 mmol) in anh. DMSO(320 mL) was added via syringe. See FIG. 17. Stirring was continued 1 hand the reaction allowed to cool. The mixture was poured into 800 mL ofice-1N hydrochloric acid and then extracted three times with 400 mLaliquots of ether. The combined organic extracts were washed with 350 mLof saturated sodium bicarbonate +400 mL of brine, dried over sodiumsulfate, and flash filtered through a 58 mm high ×84 mm dia. column ofsilica gel (200-40 mesh). Product continued eluting with additionalether. Concentration of appropriate fractions under reduced pressure andthree-fold recrystallization from aqueous ethanol gave very fine whiteneedles (11.47 g, 42.73 mmol, 76%), m.p. 134-136° C., homogeneous to TLC(20% ethyl acetate/hexanes on silica gel, R_(f) 0.45). (NA-1994A-242B)

Example 41--17-Methylenestra-1,3,5(10)-trien-3-yl acetate, 11

A solution of 17-methylenestra-1,3,5(10)-trien-3-ol (10, 5.84 g, 21.8mmol) in anh. pyridine (32 mL, 0.40 mol) and acetic anhydride (9.7 mL,0.10 mol) was stirred 24, after which ethyl acetate (250 mL) was added.See FIG. 17. The mixture was washed with three 100 mL portions of 1Nhydrochloric acid +100 mL of saturated sodium bicarbonate +100 mL ofsaturated copper sulfate +100 mL of brine, dried over magnesium sulfate,and filtered through diatomaceous earth. The residue was washed with 25mL of ethyl acetate and the combined filtrates were concentrated underreduced pressure. Recrystallization from aqueous ethanol gave lustrouswhite platelets (5.84 g, 18.8 mmol), m.p. 77-79° C. (NA-1994A-248B)

Example 42--17-Methylenestra-1,3,5(10)-trien-6-on-3-yl acetate, 12

To a suspension of chromium trioxide (6.19 g. 61.9 mmol) cooled to -8°C. (ice-salt bath) in methylene chloride (100 mL) was added2,4-dimethylpyrazole (5.95 g, 61.9 mmol). See FIG. 17. After stirring 20min., a solution of 17-methylenestra-1,3,5(10)-trien-3-yl acetate (11,2.0001 g, 6.4428 mmol) in 10 mL of chilled methylene chloride was addedover a period of 2 min. so that the temperature did not reach -6° C.Stirring was continued 2 h and the mixture was then passed through acolumn of 100 g of silica gel (200-400 mesh). Product continued elutingwith further methylene chloride. Pooling and concentration ofappropriate fractions under reduced pressure gave crude product, whichwas further purified by two-fold recrystallization from aqueous ethanolto give lustrous off-white crystals (334.0 mg, 1.030 mmol, 16%), m.p.91-94° C. TLC (25% ethyl acetate/hexanes on silica gel) showed product(R_(f) 0.47) with two minor contaminants at Rf 0.30 and 0.39.(NA-1994A-272A)

Example 43--17-Methylenestra-1,3,5(10)-trien-3,6B-diol, 13

To a suspension of lithium aluminum hydride (53.6 mg, 1.41 mmol) in anh.THF (3.0 mL) under argon cooled in a dry ice/acetone bath was added17-methylenestra-1,3,5(10)-trien-6-on-3-yl acetate (12, 251.7 mg, 0.7758mmol) in anh. THF (3.0 mL) dropwise with stirring over 8 min. See FIG.17. After stirring 2 h, the bath was removed and stirring continued afurther hour. The reaction was quenched by stirring 1/2 h with Glauber'ssalt (1.78 g). The resulting mixture was applied to a short pad ofdiatomaceous earth and extracted four times with 10 mL portions of ethylacetate. Continued extraction with five 10 mL portions of hot ethylacetate and concentration of all extracts under reduced pressure gave acolorless film. Preparative TLC (40% ethyl acetate/hexanes on silica gelGF, ¹⁰⁰⁰ IL thickness) gave a white foam (15.3 mg, 53.8 ILmol, 7%). TLC(40% ethyl acetate/hexanes on silica gel) showed major (Rf 0.29) andminor components (Rf 0.37). (NA-1994A-283B)

Example 44--17-Methylenestra-1,3,5(10)-trien-3-yl methyl ether, 14

To a stirred suspension of 17-methylenestra-1,3,5(10)-trien-3-ol (5.37g, 20.0 mmol) and potassium carbonate (50.82 g, 0.3678 mol) at reflux in90% ethanol (500 mL) was added dimethyl sulfate (5.0 mL, 53 mmol). After1/2 h reflux additional dimethyl sulfate (36 mL, 0.38 mol, in three 12mL aliquots) was added over the period of 1 h. See FIG. 17. The reactionwas refluxed a further hour, following which 360 mL of water were addedand the mixture was placed in the refrigerator overnight. The resultingsuspension was filtered and washed with 80 mL of 60% methanol +three 80mL portions of 5% (w/w) sodium hydroxide +three 80 mL portions of water.The residue was recrystallized from aqueous methanol to give whitecrystals (3.88 g, 13.7 mmol, 69%), m.p. 59-62° C. TLC (20% ethylacetate/hexanes on silica gel) showed product (R_(f) 0.63) with tracecontaminants at R_(f) 0.37 and at the origin. (NA-1994A-247)

Example 45--17-Methylenestra-2,5(10)-dien-3-yl methyl ether, 15

Approximately 70 mL of anh. ammonia was distilled through a KOH towerinto a 250 mL flame-dried 3-neck flask fitted with an inlet adapter,magnetic stirring bar, dry ice/acetone condenser, and ground glassstopper. See FIG. 17. A solution of17-methylenestra-1,3,5(10)-trien-3-yl methyl ether (14, 1.1297 g, 4.0001mmol) and t-butyl alcohol (13.21 g, 0.1782 mol) in dry THF (17 mL) wasadded, followed by lithium wire (0.47 g, 68 mg-atom) cut in smallpieces. After refluxing under argon for 6 h anh. methanol (6.6 mL) wasadded and the suspension was stirred overnight while allowing ammonia toboil off. Water (100 mL) was added and the suspension was extractedthree times with 50 mL portions of methylene chloride. The combinedorganic extracts were washed with 100 mL of brine, dried over sodiumsulfate, and filtered. The residue was washed with 25 mL of methylenechloride and the combined filtrates were concentrated under reducedpressure. The resulting light yellow oil was crystallized from aqueousethanol to give lustrous white crystals (815.0 mg, 2.865 mmol, 72%),m.p. 77-78° C., homogeneous to TLC (R_(f) 0.60, 10% ethylacetate/hexanes on silica gel). (NA-1994A-257)

Example 46--17-Methylenestr-4-en-3-one, 16

Con. hydrochloric acid (6.0 mL) and water (6.0 mL) were added to asolution of 17-methylenestra-2,5(10)-dien-3-yl methyl ether (15, 702.8mg. 2.471 mmol) in methanol (6 mL) and acetone (20 mL). See Example 17.After stirring 1 h, sodium bicarbonate (7.50 g) was added cautiously.The mixture was concentrated under reduced pressure once effervescencehad ceased and water (50 mL) was added. The mixture was extracted threetimes with 25 mL portions of methylene chloride. The combined organicextracts were washed with 50 mL of brine, dried over magnesium sulfate,and filtered through diatomaceous earth. The residue was washed with 10mL of methylene chloride and the combined filtrates were concentratedunder reduced pressure. The product was purified by decolorization withcharcoal, flash chromatography (20% ethyl acetate/hexanes on silicagel), and recrystallization from aqueous ethanol to give a white powder(302.8 mg. 1.120 mmol, 45%), m.p. 83-89° C. (NA-1994A-271)

Example 47--17-Methylenestr-4-en-3β-ol, 17

Lithium tri-t-butoxyaluminohydride (766.6 mg, 3.015 mmol) was added to asolution of 17-methylenestr-4-en-3-one (16, 203.7 mg, 0.7533 mmol) in 10mL of anh. ether and the reaction was stirred 4 h. See FIG. 18.Glauber's salt (3.80 g) was added and the suspension was stirred anadditional 1/2 h. The mixture was filtered through diatomaceous earthand the residue was washed five times with 10 mL portions of ether. Thecombined filtrates were concentrated under reduced pressure and thensubjected to preparative TLC (5% ethyl acetate/methylene chloride onsilica gel GF, 1000μ thickness) to give white needles (60.2 mg. 0.221mmol, 29%) homogeneous to TLC (Rf 0.37, 5% ethyl acetate/methylenechloride on silica gel). (NA-1994A-282)

Example 48--17-Methylenestra-1,3,5(10),7-tetraen-3-ol, 18

Methyltriphenylphosphonium bromide (1.9967 g, 5.5892 mmol) and potassiumt-butoxide (627.2 mg, 5.589 mmol) suspended in 6.1 mL of anh. DMSO underargon were lh in an oil bath (71-83° C., after which equilin (300.0 mg,1.118 mmol) in 6.1 mL of anh. DMSO was added via syringe. See FIG. 18.After stirring a further 70 min., the reaction mixture was poured into40 mL of ice water and extracted three times with 25 mL portions ofether. The combined organic extracts were washed with 25 mL of brine,dried over magnesium sulfate, and filtered through diatomaceous earth.The residue was washed with 10 mL of ether and the combined filtrateswere concentrated under reduced pressure. Flash chromatography (15%ethyl acetate/hexanes on silica gel) followed by preparative TLC (20%ethyl acetate/hexanes on silica gel GF, 1000μ thickness) gave an opaquewhite film (162.3 mg, 0.6093 :mmol, 54%). (NA)-1994A-258)

Example 49--17-Methylenestra-1,3,5(10),7-tetraen-3-yl acetate, 19

Methyltriphenylphosphonium bromide (3.33 g, 9.32 mmol) and potassiumt-butoxide (1.05 g, 9.36 mmol) suspended in 10 mL of anh. DMSO underargon was stirred 1 h in an oil bath (77-79° C.), following whichequilin (500.0 mg, 1.863 mmol) in 10 mL of anh. DMSO was added viasyringe. See FIG. 18. After stirring a further hour the cooled reactionmixture was poured into 50 mL of ice-1N hydrochloric acid and extractedthree times with 25 mL portions of ether. The combined organic extractswere washed with 25 mL of brine, dried over magnesium sulfate, andfiltered through diatomaceous earth. The residue was washed with 10 mLof ether and combined filtrates concentrated under reduced pressure. Theresulting light yellow syrup was taken up in anh. pyridine (6.3 mL, 78mmol), acetic anhydride was added (0.88 mL, 9.3 mmol), and the reactionmixture was stirred 16 h. The mixture was then poured into 100 mL of 1Nhydrochloric acid and extracted three times with 50 mL portions ofether. The combined organic extracts were washed with 100 mL ofsaturated sodium bicarbonate +100 mL of brine, dried over magnesiumsulfate, and filtered through diatomaceous earth. The residue was washedwith 25 mL of ether and the combined filtrates were concentrated underreduced pressure. The crude acetate was flash chromatographed (5% ethylacetate/hexanes on silica gel) to give a yellow resin (494.7 mg, 1,604mmol, 86%). (NA- 1994A-305)

Example 50--Estra-4,16-dien-10β-ol-3-one, 2

To a frozen (dry ice/acetone) suspension of estra-5(10),16-dien-3-one(1, 115.7 mg, 0.4513 mmol) in chloroform (3 mL) was addedm-chloroperbenzoic acid (MCPBA, 77.4%, 420.8 mg, 1.89 mEquiv. ofperacid) suspended in ether (4.3 mL) and the mixture was stirred 2 h.See FIG. 19. The reaction was then stored in a refrigerator for 18 h,after which sodium thiosulfate pentahydrate [5% (w/w), 25 g] was added.After stirring 5 min., the mixture was extracted three times with 10 mLportions of ether. The combined organic extracts were washed with 25 mLof saturated sodium bicarbonate +25 mL of brine, dried over magnesiumsulfate, and filtered through diatomaceous earth. The residue was washedwith 10 mL of ether and the combined filtrates were concentrated underreduced pressure. TLC suggested the intermediate 5B,10β-epoxide hadundergone partial elimination. Elimination was completed by refluxingthe white crystalline residue for 1 h in 20 g of 5% (w/w) potassiumhydroxide in anh. methanol. The reaction mixture was poured into 50 mLof ice water and extracted with 50 mL of ether. The organic extract waswashed twice with 50 mL portions of water, dried over sodium sulfate,and filtered through diatomaceous earth. The residue was washed with 20mL of ether and the combined filtrates were concentrated under reducedpressure. The residue was subjected to preparative TLC (50% ethylacetate/hexanes on silica gel GF, 1000μ thickness) to give a colorlessresin (19.7 mg, 72.3 μmol, 16%). (NA- 1993B-80)

Example 51--18-Nor-17-methylestra-4,13(17)-dien-3-ol, 4

To a cooled (ice water bath) solution of18-nor-17-methylestra-4,13(17)-dien-3-one (3, 0.23 g, 0.90 mmol) in anh.methanol (2.3 mL) was added sodium borohydride (0.23 g, 6.1 mmol) andthe reaction was stirred for 2 h. See FIG. 19. Solvent was removed underreduced pressure and 10 mL of water were added to the residue. Themixture was then extracted three times with 10 mL portions of methylenechloride. The combined organic extracts were washed with 10 mL of brine,dried over magnesium sulfate, and filtered through diatomaceous earth.The residue was washed twice with 5 mL of methylene chloride and thecombined filtrates were concentrated under reduced pressure. Theresulting slightly yellow solid was purified by preparative TLC (5%ethyl acetate/methylene chloride on silica gel GF, 1000μ thickness) togive a yellow solid (53.6 mg, 0.207 mmol, 23%) homogeneous to TLC (5%ethyl acetate/methylene chloride on silica gel; R_(f) 0.32). (NA-1993B-120)

Example 52--Electrophysiology of Estrene Stimulation of the Human VNOand Olfactory Epithelium

A non-invasive method has been employed to record local electricalpotentials from the human vomeronasal organ (VNO) and from the olfactoryepithelium (OE). Localized gaseous stimulation was applied to both nasalstructures at different: instances using specially designedcatheter/electrodes connected to a multichannel drug delivery system.The local response of the VNO and the OE showed a correlation with theconcentration of the ligand stimulus.

The study was performed on ten clinically normal (screened)volunteers--2 males and 8 females, ranging in age from 18 to 85 years.The studies were conducted without general or local anesthetics.

The catheter/electrodes were designed to deliver a localized stimulusand simultaneously record the response. In the case of VNO recording,the right nasal fosa of the subject was explored using a nasoscope(nasal specula) and the vomeronasal opening was localized close to theintersection of the anterior edge of the vomer and the nasal floor. Thecatheter/electrode was gently driven through the VNO-opening and theelectrode tip placed in the organ's lumen at 1 to 3 mm from the opening.The nasoscope was then removed. In the case of the OE, recording theprocedure was similar except the positioning of the catheter/electrodewas gently placed deep in the lateral part of the medial nasal duct,reaching the olfactory mucosa.

Localized gaseous stimulation was done through the catheter/electrode. Aconstant stream of clean, nonodorous, humidified air at room temperaturewas continuously passed through a channel of the stimulating system. Thestimulating ligand substances were diluted in propylene glycol, mixedwith the humidified air, and puffed for from 1 to 2 seconds through thecatheter/electrode. It is estimated that this administration providesabout 25 pg of steroid-ligand to the nasal cavity.

The results of this study are presented in FIG. 2. The response ismeasured in millivolt-seconds (mV x s). 1,3,5(10),16-Estratetraen-3-olelicits a significantly stronger VNO response in males than do the othercompounds tested (FIG. 2A). 1,3,5(10)-Estratrien-3, 16α,17β-triol alsoelicits a strong VNO response. Furthermore, the VNO response to thesetwo Estrenes is sexually dimorphic--approximately four times as strongin males as it is in females (FIG. 2B). In contrast, the OE response inboth males and females is low compared to a strong odorant such as clove(FIG. 2C).

Example 53--Measurement of the Change in Receptor Potential of theNeuroepithelium of the VNO in Response to Various steroids

The change in receptor potential in response to seven different ligandswas measured in 40 female (FIG. 3A) and 40 male (FIG. 3B) subjects. Eachsubject was administered 60 pg of each of seven substances as indicatedin the Figures. The substances were administered, each separately for 1second, using the procedure described in Example 20. The change inpotential of the neuroepithelium of VNO was recorded over time and theintegral of the change in potential for each of the forty subjects wasaveraged. The results are shown in the figure. Comparison of FIGS. 3Aand 3B show that each steroid is sexually dimorphic in its activity andthat some ligand substances are stronger in males while others arestronger in females.

Example 54--Measurement of Autonomic Responses to Estrene Stimulation ofthe VNO

Various autonomic parameters were monitored while 1,3,5(10),16-Estratetraen-3-yl-acetate was administered to 40 male subjects usingthe procedure described in Example 20. Propylene glycol was alsoadministered as a control. The ligand was administered as a 1 secondpulse. The change in autonomic function was first noted within 2 secondsand lasted for up to 45 seconds. As shown in FIG. 4, when compared to apropylene glycol control, the Estrene, induced a significant change inthe integrated receptor potential in the VNO (4A), galvanic skinresponse (4B), and skin temperature (4C).

Example 55--Comparison of the Change in Receptor Potential Induced byTwo Estrene Steroids

60 picograms of each steroid and of a propylene glycol control wereadministered to a male subject as described in Example 21. As shown inFIG. 5, 1, 3 ,5 (10),16-Estratetraen-3-ol methyl ether induced a greaterchange in receptor potential than did 1,3,5(10),16-Estratetraen-3-ylacetate.

Example 56--Psychophysiological Effect of Estrene Stimulation of the VNO

The psychophysiological effect of Estrene stimulation of the VNO ismeasured by the coordinate administration of pheromone and questionnaireevaluation of the subject before and after administration. Thequestionnaire includes a panel of adjectives used as part of thestandard Derogatis Sexual Inventory evaluation.

40 subjects, all in good health, are randomly assigned--20 exposed toplacebo and 20 exposed to about 20 picograms of1,3,5(10),16-Estratetraen-3-ol, administered as described in Example 3,supra. Subjects are given a 70 item questionnaire evaluating feelingstates immediately before and 30 minutes after administration of eitherplacebo or experimental substance. The 70 adjectives of thequestionnaire are randomly administered and subsequently clustered forevaluation based on their relevance to each mood, feeling, or charactertrait.

Example 57--Electrophysiological Studies

The following electrophysiological studies were performed in 60clinically normal human volunteers of both sexes (30 male and 30 female)whose ages ranged from 20 to 45 years. No anesthetics were used, andfemale subjects were excluded if pregnant.

The stimulation and recording system consists of a "multifunctionalminiprobe" described elsewhere (Monti-Bloch, L. and Grosser, B.1. (1991)"Effect of putative pheromones on the electrical activity of the humanvomeronasal organ and olfactory epithelium," J. Steroid Biochem. Molec.Biol. 39:573-582.). The recording electrode is a 0.3 mm silver ballattached to a small (0.1 mm) silver wire insulated with Teflon® thesurface of the electrode is first treated to produce a silver chlorideinterface, and is then covered with gelatin It is positioned within asmall caliber Teflon® catheter (dia=5 mm) such that the tip of theelectrode protrudes approximately 2 mm. The Teflon® catheter is 10 cm inlength and constitutes the terminal extension for a multichanneldelivery system which delivers a continuous air stream carrying discreetpulses of chemosensory stimuli. The air stream first passes into a smallchamber and is bubbled through a solution containing either avomeropherin or an olfactant in a diluent or the diluent alone. Asolenoid is used to rapidly redirect the air stream from the chamber toa route which bypasses the chamber. This creates a discreet pulse ofstimulant in the air stream. A second, outer Teflon® tube with adiameter of 2 mm surrounds the catheter-electrode assemblage, and itscentral end is connected to an aspirator that provides continuoussuction of 3 ml/s. This concentric arrangement of the outer suction tubeallows the emitted chemosensory stimuli to be localized to an area wecall a "minifield" (approx. dia=1 mm), and it avoids diffusion ofsubstances either to the area outside the intended stimulation site orinto the respiratory system. The entire stimulating and recordingassemblage may be positioned either on the neurosensory epitheliumwithin the VNO, or on the surface of the olfactory or respiratoryepithelium.

Electro-vomeronasogram (EVG)

Recordings are carried out in a quiet room with the subject supine; themulti-functional miniprobe is initially stabilized within the nasalcavity using a nasal retractor placed in the vestibule. Reference andground electrodes consist of silver discs (8 mm), both of which arepositioned on the glabella.

The entrance to the VNO, or vomeronasal pit, is identified by firstdilating the nasal aperture and vestibule. A 6× magnifying binocularloupe with halogen illumination is then used to introduce the tip of theTeflon® catheter and recording electrode assemblage into the VNO openingwhere it is stabilized at an approximate depth of 1 mm within thevomeronasal passage. Optimal placement of the recording electrode issignaled after testing for an adequate depolarization in response to atest substance.

Electrical signals from the recording electrode are fed to a DCamplifier after which they are digitized, computer monitored, andstored. The peak-to-peak amplitude of the signals is measured, and thearea under the depolarization wave is integrated, while continuouslymonitoring the signal both on the computer screen and on a digitaloscilloscope. Artifacts produced by respiratory movements are deleted bytraining the subjects to practice mouth breathing with velopharyngealclosure.

Chemosensory Stimulants

Olfactory test substances are cineole, and 1-carvone; vomeropherins areA, B, C, E and F. (Vomeropherins were supplied by Pherin Corporation,Menlo Park, Calif.), Samples of vomeropherins In concentration of 25-800fmoles are delivered in the continuous air stream for durations from 300milliseconds to 1 second. Usually, intervals of 3 to 5 minutes separatedeach series of short test pulses. All components of the lines carryingthe test stimuli are made of Teflon®, glass or stainless steel and arecarefully cleaned and sterilized before each use.

Electro-olfactgram (EOG)

Olfactory recordings employed the same stimulating and recordingmultifunctional miniprobe as that used for the VNO. The tip was slowlyintroduced until the recording electrode touched the olfactory mucosa.Adequate placement was signaled by a depolarization in response to apulse of the odorant test substance.

Cortical evoked activity was induced by VNO stimulation withvomeropherins, and olfactory stimulation with odorants delivered in 300ms air pulses. It was recorded using standard electroencephalographic(EEG) electrodes placed at positions Cz-Al and Tz-Al of theinternational 10120 system; the ground electrode was placed on themastoid process. Electrodermal activity (EDA) was recorded usingstandard 8 mm silver electrodes in contact with palmar skin of themedial and ring fingers respectively, through a conductive gelinterface. Skin temperature (ST) was recorded by a small (1.0 mm)thermistor probe placed in the right ear lobe. Peripheral arterial pulse(PAP) was monitored with a plethysmograph attached to the tip of theindex finger. Respiratory frequency (RF) was measured with an adjustablestrain gauge placed around the lower thorax. All electrical signals wereDC amplified, digitized (MP-100, Biopac Systems) and continuouslymonitored utilizing a computer.

Statistical Analysis

EVGs or EOGS, peak-to-peak changes and frequency changes of otherparameters were measured and statistically analyzed. The significance ofthe results was determined by either using paired t-tests or analysis ofvariance (ANOVA).

Effect of Vomeropherins on the EVG

Each of the vomeropherins was found to produce a sexually dimorphicreceptor potential (FIG. 6A-B). Recordings of the EVG were performed on30 men and 30 women (ages 20 to 45). Vomeropherins were diluted andapplied as 1 second pulses to the VNO with b minute intervals betweenpulses when questioned, the subjects were not able to "smell" orotherwise consciously detect any of the vomeropherins. This finding isin agreement with results previously reported (Monti-Bloch, L. andGrosser, B.1. (1991) "Effect of putative pheromones on the electricalactivity of the human vomeronasal organ and olfactory epithelium," J.Steroid Biochem. Molec. Biol. 39:573-582.) which indicated that neitherolfactory nor vomeropherin test stimuli delivered to the VNO elicit aperceptible sensation at the delivered concentration.

FIG. 6A shows the average response of male subjects (ages 20 to 38) tothe diluent, and to equimolar quantities (100 fmoles) of fivevomeropherins (A, B, C, D , and F), and to E, a stereoisomer of F. Theprofile of the response to each of the substances was similar in allsubjects regardless of age, and no significant differences were revealedeither by t-tests or by analysis of variance. For example, A, C and Dproduced significant effects (M₁₅ =11.4 mV, SD=3.6 mV; M₇₆ =6.4 mV, SD2.5 mV, and M₈₄ =15.1 mV, SD=4 9mV; p<0.01), that were consistent in allindividual cases. Other vomeropherins depolarized the VNO-receptors to amuch lesser extent, but with consistent mean response amplitudes fromindividual to individual. Vomeropherins active in male subjects producedlarger responses than the diluent (p<0.001). B, F and similarconcentrations of olfactants induced significantly reduced responses inthe male VNO (FIG. 6A and FIG. 7).

A similar experimental protocol was followed with the 30 female subjects(ages 20-45). Among the vomeropherins, F (100 fmoles) produced the mostsignificant differences within the group (FIG. 6B). Here, A induced asmall effect that was significantly different from F (p<0.01). In bothpopulations of subjects, active vomeropherins induced receptor responseshaving large standard deviations (FIG. 6). When the frequencydistribution of the effects of A and F was studied in males and femalesrespectively, we found a bimodal distribution. The significance of thisobservation is being studied at this point.

E, a stereoisomer of F, does not stimulate the VNO in female subjectswhile F does (FIG. 6B). This is a demonstration of the specificity ofVNO recognition of vomeropherins. In this regard it is interesting tonote that while F is a superior vomeropherin, E generates a strongerolfactory effect than does F (FIG. 6B and FIG. 7).

Effects of Vomeropherins on the EOG

The summated receptor potential from the olfactory epithelium (OE) wasrecorded in 20 subjects: 10 males and 10 females. In contrast to thesensitivity of the VNO to vomeropherins, the OE is less sensitive tothese substances. This is true for both males and females (FIG. 7A). Themean receptor potential amplitude ranged from 2.3 mV to 0.78 mV. In thisstudy, B was the only vomeropherin having significant effect in the OE(p<0.02). Of the subjects questioned about odorant sensations followingeach stimulus presentation, 16 reported no olfactory sensation, whilethree males and one female described B as an unpleasant odor. Thisfinding reveals that at the concentrations used in our study, mostvomeropherins are not effective stimulants of the olfactory receptors,but do have a clear effect on vomeronasal receptors.

Effects of Olfactants on the EVG and EOG

In contrast to vomeropherins, the olfactants 1-carvone and cineoleproduce only a minor local response in the VNO (FIG. 7B). This was truefor both men and women. As expected, these olfactants produced a strongresponse in both men and women (p<0.01) when locally applied to the OE(FIG. 7A). The diluent depolarized olfactory receptors to a lesserextent than cineole or I-carvorn (p<0.01), and it did not produce anolfactory sensation.

Reflex Effects of Vomeropherins

Studies were conducted to determine the central nervous system (CNS)reflex responses to vomeropherin stimulation of the VNO. The sexuallydimorphic local responses induced by vomeropherins (FIG. 6A and B) weremirrored in the autonomic response of male & female subjects. In malesubjects (FIG. 6C), A and C decreased skin resistance (electrodermalacuity EDA) (p<0.01, n=30). In female subjects. (FIG. 6B), F and Bproduced greater decrease in EDA than A or C (p<0.01, n=30).

Vomeropherins A and C induced a significant increase in skin temperature(ST) (FIG. 6G) in 30 male subjects (p<0.01); however D inducedsignificant temperature decrease (p<0.01). In 30 female subjects (FIG.6H) B and F evoked a significant increase in skin temperature (ST)(p<0.01) compared to A and C. In female subjects vomeropherins producedchanges in EDA and ST with a greater standard deviation than in males.

Cortical activity was recorded from Cz and Tz in male and femalesubjects during application to the VNO of air pulses (300 ms to 1 sec)containing 200 fmoles of vomeropherin (FIG. 6G and H). In males (FIG.6E) A, C and D significantly increased alpha cortical activity with alatency of 270-380 ms. D and A evoked the strongest effect (p<0.01).Synchronization of the EEG was sustained for 1.5 to 2.7 minutes afterapplication of a single pulse of active substance. In females (FIG. 6F),a single pulse (200 fmoles) of B or F applied to the VNO increased alphacortical independent of the response of olfactory receptors. We foundcharacteristic specificities in the response of the human VNO and theolfactory epithelium which suggests that they are independent functionalsystems with separate connections to the CNS (Brookover, C. (1914) Thenervus terminalis in adult man. J. Comp. Neurol. 24:131-135.) There isalso preliminary evidence that the EVG is not associated with trigeminalnociceptor endings since application of a local anesthetic (2%lidocaine) to the respiratory epithelium of the nasal septum neitherblocks nor diminishes the EVG (Monti-Bloch, L. and Grosser, B.1. (1991)"Effect of putative pheromones on the electrical activity of the humanvomeronasal organ and olfactory epithelium," J. Steroid Biochem. Molec.Biol. 39:573-582.), also, subjects failed to report sensations of painas a consequence of any of the stimulation procedures.

Additional EVG, GSR, ST and EEG tests were performed on 13 estranes inmen and women identified in Chart 1 and FIG. 20. The steroid E12/N1exhibited the best EEG-alpha activity in men. The steroid E8/N1exhibited the best EEG-beta activity in men. The steroid E7/N1 exhibitedthe best EEG-alpha activity in women. Additionally, it is noted thatE7/N1 exhibited excellent organ response, as shown by the EVG data, inboth men (FIG. 21A) and women (FIG. 20A), but there were genderdifferences in the CNS (high EEG-alpha in women, high EEG-beta in men,FIGS. 31A and B).

VNO receptors are clearly more sensitive to vomeropherins than to any ofthe olfactants tested; the opposite is true for olfactory receptors.While the OE may have receptor sites for some vomeropherins, theresponse specificity of the VNO is clearly different.

Sexual differences were noted in this specificities and effects of twogroups of vomeropherins, A, C and D; and B and F. This suggests apossible receptor-related sexual dimorphism. The findings suggest theactivation of components of the autonomic nervous system In the adulthuman by vomeropherin stimulation of the VNO.

Furthermore, the results suggest that stimulation of the VNO withvomeropherins produces synchronization of the EEG (FIG. 6G and H) Thus,the evidence herein indicates that the vomeronasal system responds to avariety of chemosensory stimuli, and that some are able to induce reflexautonomic activity.

We claim:
 1. A steroid compound of the formula: ##STR53## wherein R₁ isselected from the group consisting of one or two hydrogen atoms and oneor two halo atoms; R₂ is absent or is selected from the group consistingof hydrogen and methyl; R₃ is selected from the group consisting ofhydroxy, lower alkoxy, lower acyloxy, benzoyl, cypionyl, glucouronideand sulfonyl; R₄ is selected from the group consisting of hydrogen,hydroxy, lower alkoxy, lower acyloxy, oxo and halo; R₆ s a hydrogen or ahalo; and "c", and "d" are each double bonds; and "e", "f", "g", "h",and "i" are each optional double bonds; with the proviso that:(I) whenR₃ is methoxy, then at least "e" or "f" must be present.
 2. A compoundwhich is selected from the group consisting ofESTRA-1,3,5(10),6,16-PENTAEN-3-YL ACETATE;ESTRA-1,3,5(10),7-TETRAEN-3-OL; ESTRA-1,3,5,7,9,16-HEXAEN-3-OL;ESTRA-1,3,5(10),6-TETRAEN-3-OL; ESTRA-1,3,5,7,9,16-HEXAEN-3-YL ACETATE;ESTRA-1,3,5(10),7,1 6-PENTAEN-3-OL; ESTRA-1,3,5(10)-7,16-PENTAEN-3-YACETATE; ESTRA-1,3,5(10),6,16-PENTAEN-3-OL.
 3. A compound which is3-METHOXYESTRA-2,5(10),16-TRIENE.
 4. A compound which is selected fromthe group consisting of 3-HYDROXYESTRA-1,3,5(10),16-TETRAEN-6-ONE;6-OXOESTRA-1,3,5(10), 16-TETRAEN-3-YL ACETATE;ESTRA-1,3,5(10),16-TETRAENE-3,6β-DIOL; 6β-HYDROXYESTRA- 1,3,5(1 0),16-TETRAEN-3-YL ACETATE; ESTRA-4,9,16-TRIEN-3-ONE; ESTRA-5( 10),16-DIEN-3α-OL; and ESTRA-5(10),16-DIEN-3β-OL.